Novel dgat genes for increased seed storage lipid production and altered fatty acid profiles in oilseed plants

ABSTRACT

Transgenic oilseeds having increased total fatty acid content of at least 10% and altered fatty acid profiles when compared to the total fatty acid content of null segregant oilseeds are described. Novel DGAT genes are used to achieve the increase in seed storage lipids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/329,939, filed Dec. 19, 2011, which is a continuation of U.S.application Ser. No. 12/470,569, now U.S. Pat. No. 8,101,819, filed May22, 2009, which claims the benefit of U.S. Provisional Application No.61/055,579, filed May 23, 2008, the contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

This invention is in the field of biotechnology, in particular, thispertains to polynucleotide sequences encoding diacylglycerolacyltransferase genes and the use of these acyltransferases forincreased seed storage lipid production and altered fatty acid profilesin oilseed plants.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named432419SEQLIST.txt, created on Apr. 24, 2013, and having a size of 850kilobytes and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Plant oil is a valuable renewable resource, with annual world productionof 145 million metric tons valued at over 80 billion U.S. dollars(Rupilius and Ahmad, 2007, Eur J Lipid Sci Technol 109:433-439). Methodsto increase the content, and to improve the composition of plant oilsare therefore desired. Plant lipids have a variety of industrial andnutritional uses and are central to plant membrane function and climaticadaptation. These lipids represent a vast array of chemical structures,and these structures determine the physiological and industrialproperties of the lipid. Many of these structures result either directlyor indirectly from metabolic processes that alter the degree ofunsaturation of the lipid. Different metabolic regimes in differentplants produce these altered lipids, and either domestication of exoticplant species or modification of agronomically adapted species isusually required to produce economically large amounts of the desiredlipid.

There are limitations to using conventional plant breeding to alterfatty acid composition and content. Plant breeding will rarely uncovermutations that a) result in a dominant (“gain-of-function”) phenotype,b) that do not have negative pleiotropic effects on growth or agronomicproperties, and c) that are in an enzyme that exerts primary controlover fatty acid levels of composition. In cases where desired phenotypesare available in mutant corn lines, their introgression into elite linesby traditional breeding techniques is slow and expensive, since thedesired oil compositions are likely the result of several recessivegenes.

Recent molecular and cellular biology techniques offer the potential forovercoming some of the limitations of the conventional breedingapproach. Some of the particularly useful technologies are seed-specificexpression of foreign genes in transgenic plants [see Goldberg et al(1989) Cell 56:149-160], and the use of antisense RNA to inhibit planttarget genes in a dominant and tissue-specific manner [see van der Krolet al (1988) Gene 72:45-50]. Other advances include the transfer offoreign genes into elite commercial varieties of commercial oilcrops,such as soybean [Chee et al (1989) Plant Physiol. 91:1212-1218; Christouet at (1989) Proc. Natl. Acad. Sci. U.S.A. 86:7500-7504; Hinchee et al(1988) Bio/Technology 6:915-922; EPO publication 0 301 749 A2], rapeseed[De Block et al (1989) Plant Physiol. 91:694-701], and sunflower[Everett et al (1987) Bio/Technology 5:1201-1204], and the use of genesas restriction fragment length polymorphism (RFLP) markers in a breedingprogram, which makes introgression of recessive traits into elite linesrapid and less expensive [Tanksley et al (1989) Bio/Technology7:257-264]. However, application of each of these technologies requiresidentification and isolation of commercially-important genes.

Most free fatty acids become esterified to coenzyme A (CoA), to yieldacyl-CoAs. These molecules are then substrates for glycerolipidsynthesis in the endoplasmic reticulum of the cell, where phosphatidicacid and diacylglycerol (DAG) are produced. Either of these metabolicintermediates may be directed to membrane phospholipids (e.g.,phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine) orDAG may be directed to form triacylglycerols (TAGs), the primary storagereserve of lipids in eukaryotic cells.

Diacylglycerol acyltransferase (“DGAT”) is an integral membrane proteinthat catalyzes the final enzymatic step in the production oftriacylglycerols in plants, fungi and mammals. This enzyme isresponsible for transferring an acyl group from acyl-coenzyme-A to thesn-3 position of 1,2-diacylglycerol (“DAG”) to form triacylglycerol(“TAG”). DGAT is associated with membrane and lipid body fractions inplants and fungi, particularly, in oilseeds where it contributes to thestorage of carbon used as energy reserves. TAG is believed to be animportant chemical for storage of energy in cells. DGAT is known toregulate TAG structure and to direct TAG synthesis. Furthermore, it isknown that the DGAT reaction is specific for oil synthesis.

TAG is the primary component of vegetable oil in plants, It is used bythe seed as a stored form of energy to be used during seed germination.

Two different families of DGAT proteins have been identified. The firstfamily of DGAT proteins (“DGAT1”) is related to the acyl-coenzymeA:cholesterol acyltransferase (“ACAT”) and has been described in U.S.Pat. Nos. 6,100,077 and 6,344,548. A second family of DGAT proteins(“DGAT2”) is unrelated to the DGAT1 family and is described in PCTPatent Publication WO 2004/011671 published Feb. 5, 2004. Otherreferences to DGAT genes and their use in plants include PCT PublicationNos. WO2004/011,671, WO1998/055,631, and WO2000/001,713, and US PatentPublication No. 20030115632.

Applicants' Assignee's copending published patent application US2006-0094088 describes genes for DGATs of plants and fungi and their useis in modifying levels of polyunsaturated fatty acids (“PUFAs”) inedible oils.

Applicants' Assignee's published PCT application WO 2005/003322describes the cloning of phosphatidylcholine diacylglycerolacyltransferase and DGAT2 for altering PUFA and oil content inoleaginous yeast.

SUMMARY OF THE INVENTION

The present invention concerns a transgenic soybean seed havingincreased total fatty acid content of at least 10% when compared to thetotal fatty acid content of a null segregant soybean seed.

In a second embodiment, the present invention concerns a method forincreasing the total fatty acid content of a soybean seed comprising:

(a) transforming at least one soybean cell with one or more recombinantconstructs having at least one novel DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% when compared to thetotal fatty acid content of a null segregant soybean seed.

In a third embodiment, the present invention concerns a transgenic cornkernel having increased total fatty acid content of at least 10% whencompared to the total fatty acid content of a null segregant cornkernel.

In a fourth embodiment, the present invention concerns a method forincreasing the total fatty acid content of a corn kernel comprising:

(a) transforming at least one corn kernel with one or more recombinantconstructs having at least one novel DGAT sequence;

(b) selecting the transformed corn kernel(s) of step (a) having anincreased total fatty acid content of at least 10% when compared to thetotal fatty acid content of a null segregant corn kernel.

In a fifth embodiment, the present invention concerns a transgenicsoybean seed having increased total fatty acid content of at least 10%and an increased oleic acid content of at least 25% when compared to thetotal fatty acid content and oleic acid content of a null segregantsoybean seed.

In a further embodiment, the present invention concerns a transgenicsoybean having increased total fatty acid content of at least 10% and atleast any one of i) an increased oleic acid content of at least 25%; ii)a decreased linolenic acid content of at least 25%; iii) a decreasedlinoleic acid content of at least 4%; iv) a decreased palmitic acidcontent of at least 8%; and v) an increased stearic acid content of atlease 14% when compared to the total fatty acid content and oleic,linolenic acid, linoelic acid, palmitic acid or stearic acid,respectively, content of a null segregant soybean seed.

In an sixth embodiment, the present invention concerns a method forincreasing the total fatty acid content and oleic acid content of asoybean seed comprising:

(a) transforming at least one soybean cell with one or more recombinantconstructs having at least one novel DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and an increasedoleic acid content of at least 25% when compared to the total fatty acidcontent and oleic acid content of a null segregant soybean seed.

In a seventh embodiment, the present invention concerns a method forincreasing the total fatty acid content and decreasing linolenic acidcontent of a soybean seed comprising:

(a) transforming at least one soybean cell with one or more recombinantconstructs having at least one novel DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and a decreasedlinolenic acid content of at least 25% when compared to the total fattyacid content and oleic acid content of a null segregant soybean seed.

In an eighth embodiment, the present invention concerns a method forincreasing the total fatty acid content and decreasing linoleic acidcontent of a soybean seed comprising:

(a) transforming at least one soybean cell with one or more recombinantconstructs having at least one novel DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and a decreasedlinoleic acid content of at least 4% when compared to the total fattyacid content and oleic acid content of a null segregant soybean seed.

In a ninth embodiment, the present invention concerns a method forincreasing the total fatty acid content and decreased palmitic acidcontent of a soybean seed comprising:

(a) transforming at least one soybean cell with one or more recombinantconstructs having at least one novel DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and a decreasedpalmitic acid content of at least 8% when compared to the total fattyacid content and oleic acid content of a null segregant soybean seed.

In a tenth embodiment, the present invention concerns a method forincreasing the total fatty acid content and stearic acid content of asoybean seed comprising:

(a) transforming at least one soybean cell with one or more recombinantconstructs having at least one novel DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and an increasedstearic acid content of at least 14% when compared to the total fattyacid content and oleic acid content of a null segregant soybean seed.

Any of the transgenic seed of the invention may comprise a recombinantconstruct having at least one DGAT sequence which can be selected fromthe group consisting of DGAT1, DGAT2 and DGAT1 in combination withDGAT2. Furthermore, the DGAT sequence can be a tree nut or shuffled DGATsequence. Furthermore, the DGAT sequence can contain amino acidsubstitutions that result in greater oil increases than are achievedwith the non-substituted sequence.

In an eleventh embodiment the present invention concerns an isolatedpolynucleotide comprising:

(a) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity wherein the polypeptide has at least 80% aminoacid identity, based on the Clustal V method of alignment, when comparedto an amino acid sequence as set forth in SEQ ID NOs:8, 10, or 12;

(b) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity, wherein the nucleotide sequence has at least80%, 85%, 90%, 95%, or 100% sequence identity, based on the BLASTNmethod of alignment, when compared to a nucleotide sequence as set forthin SEQ ID NO: 7, 9, or 11:

(c) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity, wherein the nucleotide sequence hybridizesunder stringent conditions to a nucleotide sequence as set forth in SEQID NO: 7, 9, or 11; or

(d) a complement of the nucleotide sequence of (a), (b) or (c), whereinthe complement and the nucleotide sequence consist of the same number ofnucleotides and are 100% complementary.

The isolated polynucleotide may be obtained from one or more ediblenuts, such as, but not limited to, hazelnut, hickory, pistachio, andpecan. The isolated polynucleotide may also be part of a recombinant DNAconstruct comprising at least one regulatory sequence. This recombinantconstruct may also be comprised in a cell. This cell may be from anoilseed plant. Suitable oilseed plants include, but are not limited to,soybean, corn, canola, sunflower, flax, cotton, and safflower.

In a twelfth embodiment the present invention concerns a method forincreasing the total fatty acid content of an oilseed comprising:

(a) transforming at least one oilseed cell with the above mentionedrecombinant construct;

(b) selecting the transformed oilseed cell(s) of step (a) having anincreased total fatty acid content when compared to the total fatty acidcontent of a null segregant oilseed.

Also within the scope of the invention are product(s) and/orby-product(s) obtained from the transgenic soybean seeds of theinvention.

In another aspect this invention concerns an isolated nucleic acidfragment encoding a modified Type 1 diacylglycerol acyltransferasepolypeptide such that the modified Type 1 diacylglycerol acyltransferasepolypeptide has at least one amino acid substitution selected from thegroup consisting of:

a non-alanine at a position corresponding to position 12 of SEQ ID NO:12to alanine,

a non-proline at a position corresponding to position 30 of SEQ ID NO:12to proline,

a non-alanine at a position corresponding to position 31 of SEQ ID NO:12to alanine,

a non-serine at a position corresponding to position 48 of SEQ ID NO:12to serine,

a non-serine at a position corresponding to position 49 of SEQ ID NO:12to serine,

a non-aspartate at a position corresponding to position 51 of SEQ IDNO:12 to aspartate,

a non-aspartate at a position corresponding to position 52 of SEQ IDNO:12 to aspartate,

a non-threonine at a position corresponding to position 59 of SEQ IDNO:12 to threonine,

a non-threonine at a position corresponding to position 73 of SEQ IDNO:12 to threonine,

a non-asparagine at a position corresponding to position 79 of SEQ IDNO:12 to asparagine,

a non-leucine at a position corresponding to position 118 of SEQ IDNO:12 to leucine,

a non-alanine at a position corresponding to position 123 of SEQ IDNO:12 to alanine,

a non-valine at a position corresponding to position 128 of SEQ ID NO:12to valine,

a non-leucine at a position corresponding to position 139 of SEQ IDNO:12 to leucine,

a non-isoleucine at a position corresponding to position 155 of SEQ IDNO:12 to isoleucine,

a non-alanine at a position corresponding to position 181 of SEQ IDNO:12 to alanine,

a non-serine at a position corresponding to position 184 of SEQ ID NO:12to serine,

a non-valine at a position corresponding to position 197 of SEQ ID NO:12to valine,

a non-valine at a position corresponding to position 198 of SEQ ID NO:12to valine,

a non-methionine at a position corresponding to position 205 of SEQ IDNO:12 to methionine,

a non-threonine at a position corresponding to position 211 of SEQ IDNO:12 to threonine,

a non-histidine at a position corresponding to position 218 of SEQ IDNO:12 to histidine,

a non-valine at a position corresponding to position 222 of SEQ ID NO:12to valine,

a non-lysine at a position corresponding to position 241 of SEQ ID NO:12to lysine,

a non-valine at a position corresponding to position 247 of SEQ ID NO:12to valine,

a non-valine at a position corresponding to position 251 of SEQ ID NO:12to valine,

a non-serine at a position corresponding to position 256 of SEQ ID NO:12to serine,

a non-serine at a position corresponding to position 257 of SEQ ID NO:12to serine,

a non-phenylalanine at a position corresponding to position 266 of SEQID NO:12 to phenylalanine,

a non-alanine at a position corresponding to position 267 of SEQ IDNO:12 to alanine,

a non-glutamate at a position corresponding to position 281 of SEQ IDNO:12 to glutamate,

a non-aspartate at a position corresponding to position 288 of SEQ IDNO:12 to aspartate,

a non-glutamate at a position corresponding to position 293 of SEQ IDNO:12 to glutamate,

a non-asparagine at a position corresponding to position 294 of SEQ IDNO:12 to asparagine,

a non-threonine at a position corresponding to position 299 of SEQ IDNO:12 to threonine,

a non-asparagine at a position corresponding to position 301 of SEQ IDNO:12 to asparagine,

a non-leucine at a position corresponding to position 308 of SEQ IDNO:12 to leucine,

a non-glycine at a position corresponding to position 327 of SEQ IDNO:12 to glycine,

a non-leucine at a position corresponding to position 329 of SEQ IDNO:12 to leucine,

a non-leucine at a position corresponding to position 334 of SEQ IDNO:12 to leucine,

a non-valine at a position corresponding to position 337 of SEQ ID NO:12to valine,

a non-valine at a position corresponding to position 338 of SEQ ID NO:12to valine,

a non-glutamine at a position corresponding to position 356 of SEQ IDNO:12 to glutamine,

a non-asparagine at a position corresponding to position 363 of SEQ IDNO:12 to asparagine,

a non-serine at a position corresponding to position 390 of SEQ ID NO:12to serine,

a non-valine at a position corresponding to position 399 of SEQ ID NO:12to valine,

a non-isoleucine at a position corresponding to position 436 of SEQ IDNO:12 to isoleucine,

a non-alanine at a position corresponding to position 451 of SEQ IDNO:12 to alanine,

a non-serine at a position corresponding to position 457 of SEQ ID NO:12to serine,

a non-methionine at a position corresponding to position 475 of SEQ IDNO:12 to methionine,

a non-phenylalanine at a position corresponding to position 486 of SEQID NO:12 to phenylalanine,

a non-isoleucine at a position corresponding to position 488 of SEQ IDNO:12 to isoleucine,

a non-leucine at a position corresponding to position 491 of SEQ IDNO:12 to leucine,

a non-lysine at a position corresponding to position 502 of SEQ ID NO:12to lysine,

a non-serine at a position corresponding to position 514 of SEQ ID NO:12to serine,

a non-valine at a position corresponding to position 518 of SEQ ID NO:12to valine, and

a non-valine at a position corresponding to position 531 of SEQ ID NO:12to valine, when compared to the unmodified Type 1 diacylglycerolacyltransferase polypeptide, wherein the position corresponding to aposition of SEQ ID NO: 12 is based on an alignment using Clustal V ofSEQ ID NO:12 and the unmodified Type 1 diacylglycerol acyltransferasepolypeptide.

This invention further concerns an isolated nucleic acid fragmentencoding a modified Type 1 diacylglycerol acyltransferase polypeptidesuch that the modified Type 1 diacylglycerol acyltransferase polypeptidehas at least one amino acid substitution selected from the groupconsisting of:

a non-alanine at a position corresponding to position 24 of SEQ IDNO:153 to alanine,

a non-asparagine at a position corresponding to position 58 of SEQ IDNO:153 to asparagine,

a non-alanine at a position corresponding to position 146 of SEQ IDNO:153 to alanine,

a non-methionine at a position corresponding to position 170 of SEQ IDNO:153 to methionine,

a non-lysine at a position corresponding to position 206 of SEQ IDNO:153 to lysine,

a non-valine at a position corresponding to position 216 of SEQ IDNO:153 to valine,

a non-phenylalanine at a position corresponding to position 231 of SEQID NO:153 to phenylalanine,

a non-glutamate at a position corresponding to position 258 of SEQ IDNO:153 to glutamate,

a non-threonine at a position corresponding to position 264 of SEQ IDNO:153 to threonine,

a non-leucine at a position corresponding to position 273 of SEQ IDNO:153 to leucine,

a non-leucine at a position corresponding to position 299 of SEQ IDNO:153 to leucine,

a non-valine at a position corresponding to position 303 of SEQ IDNO:153 to valine,

a non-serine at a position corresponding to position 355 of SEQ IDNO:153 to serine,

a non-valine at a position corresponding to position 364 of SEQ IDNO:153 to valine,

a non-arginine at a position corresponding to position 401 of SEQ IDNO:153 to arginine,

a non-serine at a position corresponding to position 422 of SEQ IDNO:153 to serine,

a non-methionine at a position corresponding to position 440 of SEQ IDNO:153 to methionine,

a non-lysine at a position corresponding to position 467 of SEQ IDNO:153 to lysine,

a non-serine at a position corresponding to position 479 of SEQ IDNO:153 to serine,

a non-valine at a position corresponding to position 483 of SEQ IDNO:153 to valine, when compared to the unmodified Type 1 diacylglycerolacyltransferase polypeptide, wherein the position corresponding to aposition of SEQ ID NO:153 is based on an alignment using Clustal V ofSEQ ID NO:153 and the unmodified Type 1 diacylglycerol acyltransferasepolypeptide.

The above mentioned isolated nucleic acids can be further used inmethods to increase fatty acid content of an oilseed by: 1)incorporating the isolated nucleic acid inot a recombinant DNA constructcomprising at least one regulatory element, 2) introducing therecombinant DNA construct into an oilseed cell, and selecting transgeniccells that have increased fatty acid content when compared tonon-transgenic null segregants. Plants produced by this method, andplants that incorporate the recombinant DNA construct of the invention,are also claimed, as are the progeny of those plants. Furthermore,by-product and oil products produced from these plants are also claimed.

In a final embodiment the present invention concerns fungi, or microbialoleaginous organisms, comprising a recombinant DNA construct comprisingany isolated nucleic acid fragments encoding any diacylglycerolacyltransferase of the present invention. Further, the fungal cell canbe, but is not limited to, Yarrowia, Candida, Rhodotorula,Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing, whichform a part of this application.

FIG. 1 provides plasmid map for yeast expression vector pSZ378.

FIG. 2 provides plasmid map for PHP35885, comprising novel DGATCA-DGAT1-C11 in yeast expression vector.

FIG. 3 provides oil content of soybean somatic embryos transformed withthe hazelnut DGAT gene or with four novel DGAT genes.

FIG. 4 provides plasmid map for PHP36707, comprising novel DGATCA-DGAT1-C11 in maize transformation vector.

FIG. 5 A-F provides the Clustal V alignment of 31 plant DGAT1 sequences.Certain amino acid substitution positions that give rise to higher oilcontent in yeast and plants are boxed. Sequences aligned includehazelnut DGAT1 (SEQ ID NO:12); soybean DGAT1 (SEQ ID NO:153); a soybeanshuffled (Jail Call) DGAT1 (SEQ ID NO:195); soybean DGAT1 (SEQ IDNO:16); soybean (Glycine max) GmDGAT1 (gi56199782, SEQ ID NO:196);soybean (Glycine max) GmDGAT1a (gi93204650 SEQ ID NO:197); soybean(Glycine max) GmDGAT1b (gi93204652, SEQ ID NO:198); maize (Zea mays)ZM-DGAT1 (gi187806722, SEQ ID NO:199); Arabidopsis thaliana DGAT1(gi91152592, SEQ ID NO:200); grape (Vitis vinifora) DGAT1 (gi225444869,SEQ ID NO:201); grape (Vitis vinifora) DGAT1 (gi147859067, SEQ IDNO:202); Vernonia galamensis_DGAT1 (gi157092192, SEQ ID NO:203);Vernonia galamensis DGAT1 (gi157092190, SEQ ID NO:204); tung oil tree(Vernicia fordii) DGAT1 (gi86279632, SEQ ID NO:205); nasturtiumTropaeolum majus DGAT1 (gi67043496, SEQ ID NO:206); black cottonwoodPopulus trichcarpa_DGAT1 (gi224087975, SEQ ID NO:207); beefsteak(Perilla frutescens) DGAT1 gi10803053, SEQ ID NO:208); rice (Oryzasativa)_DGAT1 (gi57231736, SEQ ID NO:209); rice (Oryza sativa) DGAT1(gi53791817, SEQ ID NO:210); rice (Oryza sativa) DGAT1 (gi51854436, SEQID NO:211); olive (Olea europaea)_DGAT1 (gi41387497, SEQ ID NO:212;Medicago truncatula DGAT1 (gi124361135, SEQ ID NO:213); Lotus japonicaDGAT1 (gi57545061, SEQ ID NO:214); Jatropha curcas DGAT1 (gi82582915,SEQ ID NO:215); burning bush Euonymus alata DGAT1 (gi118800682, SEQ IDNO:216); Brassica napus DGAT1 (gi7576941, SEQ ID NO:217); mustardBrassica juncia DGAT1 (gi63376226, SEQ ID NO:218); Arabidopsis thalianaDGAT1 (gi127266368, SEQ ID NO:219); maize (Zea mays) DGAT1 (gi187806720,SEQ ID NO:220); Brassica napus DGAT1 (gi91152589, SEQ ID NO:221);Brassica napus DGAT1 (gi91152588, SEQ ID NO:222).

FIG. 6 provides oil concentration plotted versus oleic acidconcentration for MSE2515, MSE 2516, MSE2517, MSE2518 and MSE2519 fromExample 8.

The sequence descriptions summarize the Sequences Listing attachedhereto. The Sequence Listing contains one letter codes for nucleotidesequence characters and the single and three letter codes for aminoacids as defined in the IUPAC-IUB standards described in Nucleic AcidsResearch 13:3021-3030 (1985) and in the Biochemical Journal219(2):345-373 (1984).

Summary of Nucleic Acid and Protein SEQ ID Numbers

Nucleic acid Protein Description and Abbreviation SEQ ID NO. SEQ ID NO.Hickory (Carya ovata) diacylglycerol  7  8 acyltransferase 1a(CO-DGAT1a) (1617bp) (538aa) Hickory (Carya ovata) diacylglycerol  9  10acyltransferase 1a (CO-DGAT1b) (1617bp) (538aa) Hazelnut (Corylusamericana) diacylglycerol  11  12 acyltransferase 1 (CA-DGAT1) (1620bp)(539aa) Plasmid PHP32238 comprising the hickory  13 DGAT1a (CO-DGAT1a)(9074bp) Plasmid PHP32396 comprising the hickory  14 DGAT1b (CO-DGAT1b)(9074bp) Plasmid PHP32395 comprising the hazelnut  15 DGAT1 (CA-DGAT1)(9077bp) Soybean (Glycine max) DGAT1  16 (504aa) Arabidopsis thalianaDGAT1  17 (520aa) Wheat (Triticum aestivum) DGAT1  18 (508aa) Maize (Zeamays) DGAT1  19 (494aa) Plasmid pKS394  20 (11696bp)  Plasmid pKS352  21(10866bp)  Plasmid pSZ378  22 (7457bp) Hazelnut DGAT with internal BamHIand EcoRI  23 sites removed (CA-DGAT1*) (1620bp) Plasmid pKR52  24(9065bp) Hazelnut (Corylus americana) CA-DGAT1-A1  25  26 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-A2  27  28 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-A9  29  30 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-A14  31  32 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-A15  33  34 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-A16  35  36 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-A17  37  38 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-B6  39  40 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-C1  41  42 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-C5  43  44 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-C7  45  46 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-C8  47  48 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-C9  49  50 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-C10  51  52 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-C11  53  54 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-C13  55  56 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-C15  57  58 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D2  59  60 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D4  61  62 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D5  63  64 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D6  65  66 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D7  67  68 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D8  69  70 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D9  71  72 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D10  73  74 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D12  75  76 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D14  77  78 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D15  79  80 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D16  81  82 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D17  83  84 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D18  85  86 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D19  87  88 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-D20  89  90 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E1  91  92 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E2  93  94 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E3  95  96 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E4  97  98 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E5  99 100 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E6 101 102 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E7 103 104 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E8 105 106 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E9 107 108 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E10 109 110 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E11 111 112 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E13 113 114 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E15 115 116 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E16 117 118 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-E19 119 120 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-F4 121 122 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-F8 123 124 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-F17 125 126 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-F18 127 128 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-F19 129 130 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-J1 131 132 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-J12 133 134 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-J13 135 136 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-J16 137 138 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-J21 139 140 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-J24 141 142 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-J32 143 144 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-J34 145 146 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-J37 147 148 (1620bp)(539aa) Hazelnut (Corylus americana) CA-DGAT1-J38 149 150 (1620bp)(539aa) Plasmid PHP35885 151 (9065bp) Soybean (Glycine max) GM-DGAT1 152153 (1515bp) (504aa) Soybean (Glycine max) GM-DGAT1-C9 154 155 (1515bp)(504aa) Soybean (Glycine max) GM-DGAT1-C10 156 157 (1515bp) (504aa)Soybean (Glycine max) GM-DGAT1-C11 158 159 (1515bp) (504aa) Soybean(Glycine max) GM-DGAT1-C9C10C11 160 161 (1515bp) (504aa) Maize (Zeamays) ZM-DGAT1(MOD1) 162 (1485bp) Maize (Zea mays) ZM-DGAT1(MOD2) 163164 (1485bp) (494aa) Maize (Zea mays) ZM-DGAT1(MOD3) 165 166 (1485bp)(494aa) Maize (Zea mays) ZM-DGAT1(MOD4) 167 168 (1485bp) (494aa) Maize(Zea mays) ZM-DGAT1(MOD5) 169 170 (1485bp) (494aa) Plasmid PHP40102 171(8948bp) Maize-Hazelnut DGAT1 chimera 172 173 (1482bp) (493aa)Maize-Hazelnut DGAT1-C11 chimera 174 175 (1482bp) (493aa) Peptide 1 fromhazelnut DGAT 176  (15aa) Peptide 2 from maize DGAT 177  (16aa) PlasmidpKR72 178 (7085bp) Plasmid pKR1466 179 (8611bp) Plasmid pKR1515 180(8611bp) Plasmid pKR1516 181 (8611bp) Plasmid pKR1517 182 (8611bp)Plasmid pKR1520 183 (8611bp) Soybean (Glycine max) GM-DGAT1-J16 184 185(1515bp) (504aa) Soybean (Glycine max) GM-DGAT1-J24 186 187 (1515bp)(504aa) Soybean (Glycine max) GM-DGAT1-J32 188 189 (1515bp) (504aa)Soybean (Glycine max) GM-DGAT1-J37 190 191 (1515bp) (504aa) Soybean(Glycine max) GM-DGAT1- 192 193 J16J24J32J37 (1515bp) (504aa) Soybean(Glycine max) GM-DGAT1-Jall Call 194 195 (1515bp) (504aa) Soybean(Glycine max) GmDGAT1_gi56199782 196 (498aa) Soybean (Glycine max)GmDGAT1a gi93204650 197 (498aa) Soybean (Glycine max)GmDGAT1b_gi93204652 198 (504aa) Maize (Zea mays) ZM-DGAT1 gi187806722199 (494aa) Arabidopsis thaliana DGAT1_gi91152592 200 (547aa) Grape(Vitis vinifora)_ DGAT1_gi225444869 201 (518aa) Grape (Vitis vinifora)_DGAT1_gi147859067 202 (502aa) Vernonia galamensis_ DGAT1_gi157092192 203(517aa) Vernonia galamensis _ DGAT1_gi157092190 204 (523aa) Tung oiltree (Vernicia fordii)_DGAT1 gi86279632 205 (526aa) Tropaeolum majusDGAT1 gi67043496 206 (518aa) Populus trichocarpa_ DGAT1_gi224087975 207(446aa) Beefsteak (Perilla frutescens) DGAT1 gi10803053 208 (534aa) Rice(Oryza sativa)_ DGAT1_gi57231736 209 (538aa) Rice (Oryza sativa)DGAT1_(—)_gi53791817 210 (477aa) Rice (Oryza sativa)_ DGAT1_gi51854436211 (504aa) Olive (Olea europaea)_ DGAT1_gi41387497 212 (532aa) Medicagotruncatula_ DGAT1_gi124361135 213 (539aa) Lotus japonicaDGAT1_(—)_gi57545061 214 (511aa) Jatropha curcas_ DGAT1_gi82582915 215(521aa) Euonymus alata_ DGAT1_gi118800682 216 (507aa) Brassica napus_DGAT1_gi7576941 217 (501aa) Brassica juncia_ DGAT1_gi63376226 218(503aa) Arabidopsis thaliana_ DGAT1 gi127266368 219 (520aa) Maize (Zeamays) DGAT1_gi187806720 220 (494aa) Brassica napus_ DGAT1_gi91152589 221(503aa) Brassica napus_ DGAT1_gi91152588 222 (341aa)

SEQ ID NOs:1-6 correspond to primers (p21, p18, p33, p34, p37b, and p38,respectively) used to PCR amplify the two hickory (Carya ovata) and onehazelnut (Corylus americana) DGAT1 genes.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

In the context of this disclosure, a number of terms and abbreviationsare used. The following definitions are provided.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“American Type Culture Collection” is abbreviated ATCC.

Acyl-CoA:sterol-acyltransferase” is abbreviated ARE2.

“Phospholipid:diacylglycerol acyltransferase” is abbreviated PDAT.

“Diacylglycerol acyltransferase” is abbreviated DAG AT or DGAT.

“Diacylglycerol” is abbreviated DAG.

“Triacylglycerols” are abbreviated TAGs.

“Co-enzyme A” is abbreviated CoA.

The term “fatty acids” refers to long chain aliphatic acids (alkanoicacids) of varying chain length, from about C₁₂ to C₂₂ (although bothlonger and shorter chain-length acids are known). The predominant chainlengths are between C₁₆ and C₂₂. The structure of a fatty acid isrepresented by a simple notation system of “X:Y”, where X is the totalnumber of carbon (C) atoms in the particular fatty acid and Y is thenumber of double bonds.

Generally, fatty acids are classified as saturated or unsaturated. Theterm “saturated fatty acids” refers to those fatty acids that have no“double bonds” between their carbon backbone. In contrast, “unsaturatedfatty acids” have “double bonds” along their carbon backbones (which aremost commonly in the cis-configuration). “Monounsaturated fatty acids”have only one “double bond” along the carbon backbone (e.g., usuallybetween the 9^(th) and 10^(th) carbon atom as for palmitoleic acid(16:1) and oleic acid (18:1)), while “polyunsaturated fatty acids” (or“PUFAs”) have at least two double bonds along the carbon backbone (e.g.,between the 9^(th) and 10^(th), and 12^(th) and 13^(th) carbon atoms forlinoleic acid (18:2); and between the 9^(th) and 10^(th), 12^(th) and13^(th), and 15^(th) and 16^(th) for α-linolenic acid (18:3)).

“Microbial oils” or “single cell oils” are those oils naturally producedby microorganisms (e.g., algae, oleaginous yeasts and filamentous fungi)during their lifespan. The term “oil” refers to a lipid substance thatis liquid at 25° C. and usually polyunsaturated. In contrast, the term“fat” refers to a lipid substance that is solid at 25° C. and usuallysaturated.

“Lipid bodies” refer to lipid droplets that usually are bounded byspecific proteins and a monolayer of phospholipid. These organelles aresites where most organisms transport/store neutral lipids. Lipid bodiesare thought to arise from microdomains of the endoplasmic reticulum thatcontain TAG-biosynthesis enzymes; and, their synthesis and size appearto be controlled by specific protein components.

“Neutral lipids” refer to those lipids commonly found in cells in lipidbodies as storage fats and oils and are so called because at cellularpH, the lipids bear no charged groups. Generally, they are completelynon-polar with no affinity for water. Neutral lipids generally refer tomono-, di-, and/or triesters of glycerol with fatty acids, also calledmonoacylglycerol, diacylglycerol or TAG, respectively (or collectively,acylglycerols). A hydrolysis reaction must occur to release free fattyacids from acylglycerols.

The terms “triacylglycerol”, “oil” and “TAGs” refer to neutral lipidscomposed of three fatty acyl residues esterified to a glycerol molecule(and such terms will be used interchangeably throughout the presentdisclosure herein). Such oils can contain long chain PUFAs, as well asshorter saturated and unsaturated fatty acids and longer chain saturatedfatty acids. Thus, “oil biosynthesis” generically refers to thesynthesis of TAGs in the cell.

The term “DAG AT” or “DGAT” refers to a diacylglycerol acyltransferase(also known as an acyl-CoA-diacylglycerol acyltransferase or adiacylglycerol O-acyltransferase) (EC 2.3.1.20). This enzyme isresponsible for the conversion of acyl-CoA and 1,2-diacylglycerol to TAGand CoA (thereby involved in the terminal step of TAG biosynthesis). Twofamilies of DAG AT enzymes exist: DGAT1 and DGAT2. The former familyshares homology with the acyl-CoA:cholesterol acyltransferase (ACAT)gene family, while the latter family is unrelated (Lardizabal et al., J.Biol. Chem. 276(42):38862-28869 (2001)).

The term “PDAT” refers to a phospholipid:diacylglycerol acyltransferaseenzyme (EC 2.3.1.158). This enzyme is responsible for the transfer of anacyl group from the sn-2 position of a phospholipid to the sn-3 positionof 1,2-diacylglycerol, thus resulting in lysophospholipid and TAG(thereby involved in the terminal step of TAG biosynthesis). This enzymediffers from DGAT (EC 2.3.1.20) by synthesizing TAG via anacyl-CoA-independent mechanism.

The term “ARE2” refers to an acyl-CoA:sterol-acyltransferase enzyme (EC2.3.1.26; also known as a sterol-ester synthase 2 enzyme), catalyzingthe following reaction: acyl-CoA+cholesterol=CoA+cholesterol ester.

As used herein, “nucleic acid” means a polynucleotide and includessingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases. Nucleic acids may also include fragments andmodified nucleotides. Thus, the terms “polynucleotide”, “nucleic acidsequence”, “nucleotide sequence” or “nucleic acid fragment” are usedinterchangeably and is a polymer of RNA or DNA that is single- ordouble-stranded, optionally containing synthetic, non-natural or alterednucleotide bases. Nucleotides (usually found in their 5′-monophosphateform) are referred to by their single letter designation as follows: “A”for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” forcytidylate or deosycytidylate, “G” for guanylate or deoxyguanylate, “U”for uridlate, “T” for deosythymidylate, “R” for purines (A or G), “Y”for pyrimidiens (C or T), “K” for G or T, “H” for A or C or T, “F′ forinosine, and “N” for any nucleotide.

The terms “subfragment that is functionally equivalent” and“functionally equivalent subfragment” are used interchangeably herein.These terms refer to a portion or subsequence of an isolated nucleicacid fragment in which the ability to alter gene expression or produce acertain phenotype is retained whether or not the fragment or subfragmentencodes an active enzyme. For example, the fragment or subfragment canbe used in the design of chimeric genes to produce the desired phenotypein a transformed plant. Chimeric genes can be designed for use insuppression by linking a nucleic acid fragment or subfragment thereof,whether or not it encodes an active enzyme, in the sense or antisenseorientation relative to a plant promoter sequence.

The term “conserved domain” or “motif” means a set of amino acidsconserved at specific positions along an aligned sequence ofevolutionarily related proteins. While amino acids at other positionscan vary between homologous proteins, amino acids that are highlyconserved at specific positions indicate amino acids that are essentialin the structure, the stability, or the activity of a protein. Becausethey are identified by their high degree of conservation in alignedsequences of a family of protein homologues, they can be used asidentifiers, or “signatures”, to determine if a protein with a newlydetermined sequence belongs to a previously identified protein family.

The terms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Theyrefer to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate gene expression or produce a certain phenotype. These termsalso refer to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotides thatdo not substantially alter the functional properties of the resultingnucleic acid fragment relative to the initial, unmodified fragment. Itis therefore understood, as those skilled in the art will appreciate,that the invention encompasses more than the specific exemplarysequences.

Moreover, the skilled artisan recognizes that substantially similarnucleic acid sequences encompassed by this invention are also defined bytheir ability to hybridize (under moderately stringent conditions, e.g.,0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or toany portion of the nucleotide sequences disclosed herein and which arefunctionally equivalent to any of the nucleic acid sequences disclosedherein. Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 80% sequence identity, or 90% sequence identity, upto and including 100% sequence identity (i.e., fully complementary) witheach other.

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will selectivelyhybridize to its target sequence. Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth et al., Anal. Biochem. 138:267-284 (1984):T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part 1, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). Hybridization and/or washconditions can be applied for at least 10, 30, 60, 90, 120, or 240minutes.

“Sequence identity” or “identity” in the context of nucleic acid orpolypeptide sequences refers to the nucleic acid bases or amino acidresidues in two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window.

Thus, “percentage of sequence identity” refers to the value determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the results by 100 to yield the percentage of sequenceidentity. Useful examples of percent sequence identities include, butare not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%,or any integer percentage from 50% to 100%. These identities can bedetermined using any of the programs described herein.

Sequence alignments and percent identity or similarity calculations maybe determined using a variety of comparison methods designed to detecthomologous sequences including, but not limited to, the MegAlign™program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.). Within the context of this application it will beunderstood that where sequence analysis software is used for analysis,that the results of the analysis will be based on the “default values”of the program referenced, unless otherwise specified. As used herein“default values” will mean any set of values or parameters thatoriginally load with the software when first initialized.

The “Clustal V method of alignment” corresponds to the alignment methodlabeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153(1989); Higgins, D. G. et al. (1992) Comput. Appl. Biosci. 8:189-191)and found in the MegAlign™ program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). For multiple alignments,the default values correspond to GAP PENALTY=10 and GAP LENGTHPENALTY=10. Default parameters for pairwise alignments and calculationof percent identity of protein sequences using the Clustal method areKTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleicacids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 andDIAGONALS SAVED=4. After alignment of the sequences using the Clustal Vprogram, it is possible to obtain a “percent identity” by viewing the“sequence distances” table in the same program.

“BLASTN method of alignment” is an algorithm provided by the NationalCenter for Biotechnology Information (NCBI) to compare nucleotidesequences using default parameters.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, from otherspecies, wherein such polypeptides have the same or similar function oractivity. Useful examples of percent identities include, but are notlimited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or anyinteger percentage from 50% to 100%. Indeed, any integer amino acididentity from 50% to 100% may be useful in describing the presentinvention, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Also, ofinterest is any full-length or partial complement of this isolatednucleotide fragment.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure.

The term “genome” as it applies to a plant cells encompasses not onlychromosomal DNA found within the nucleus, but organelle DNA found withinsubcellular components (e.g., mitochondrial, plastid) of the cell.

A “codon-optimized gene” is a gene having its frequency of codon usagedesigned to mimic the frequency of preferred codon usage of the hostcell.

An “allele” is one of several alternative forms of a gene occupying agiven locus on a chromosome. When all the alleles present at a givenlocus on a chromosome are the same that plant is homozygous at thatlocus. If the alleles present at a given locus on a chromosome differthat plant is heterozygous at that locus.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to: promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing sites, effectorbinding sites and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence that can stimulate promoter activity, and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity. Promoters that cause agene to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”. New promoters of various typesuseful in plant cells are constantly being discovered; numerous examplesmay be found in the compilation by Okamuro, J. K., and Goldberg, R. B.Biochemistry of Plants 15:1-82 (1989).

“Translation leader sequence” refers to a polynucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D., Mol.Biotechnol. 3:225-236 (1995)).

“3′ non-coding sequences”, “transcription terminator” or “terminationsequences” refer to DNA sequences located downstream of a codingsequence and include polyadenylation recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The use of different 3′ non-codingsequences is exemplified by Ingelbrecht, I. L., et al. Plant Cell1:671-680 (1989).

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript. A RNA transcript is referred toas the mature RNA when it is a RNA sequence derived frompost-transcriptional processing of the primary transcript. “MessengerRNA” or “mRNA” refers to the RNA that is without introns and that can betranslated into protein by the cell. “cDNA” refers to a DNA that iscomplementary to, and synthesized from, a mRNA template using the enzymereverse transcriptase. The cDNA can be single-stranded or converted intodouble-stranded form using the Klenow fragment of DNA polymerase I.“Sense” RNA refers to RNA transcript that includes the mRNA and can betranslated into protein within a cell or in vitro. “Antisense RNA”refers to an RNA transcript that is complementary to all or part of atarget primary transcript or mRNA, and that blocks the expression of atarget gene (U.S. Pat. No. 5,107,065). The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to antisense RNA, ribozymeRNA, or other RNA that may not be translated but yet has an effect oncellular processes. The terms “complement” and “reverse complement” areused interchangeably herein with respect to mRNA transcripts, and aremeant to define the antisense RNA of the message.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis regulated by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of regulating the expressionof that coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of theinvention can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1989).Transformation methods are well known to those skilled in the art andare described infra.

“PCR” or “polymerase chain reaction” is a technique for the synthesis oflarge quantities of specific DNA segments and consists of a series ofrepetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.).Typically, the double-stranded DNA is heat denatured, the two primerscomplementary to the 3′ boundaries of the target segment are annealed atlow temperature and then extended at an intermediate temperature. Oneset of these three consecutive steps is referred to as a “cycle”.

The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitates transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host (i.e., to a discretenucleic acid fragment into which a nucleic acid sequence or fragment canbe moved.)

The terms “recombinant construct”, “expression construct”, “chimericconstruct”, “construct”, and “recombinant DNA construct” are usedinterchangeably herein. A recombinant construct comprises an artificialcombination of nucleic acid fragments, e.g., regulatory and codingsequences that are not found together in nature. For example, a chimericconstruct may comprise regulatory sequences and coding sequences thatare derived from different sources, or regulatory sequences and codingsequences derived from the same source, but arranged in a mannerdifferent than that found in nature. Such a construct may be used byitself or may be used in conjunction with a vector. If a vector is used,then the choice of vector is dependent upon the method that will be usedto transform host cells as is well known to those skilled in the art.For example, a plasmid vector can be used. The skilled artisan is wellaware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cellscomprising any of the isolated nucleic acid fragments of the invention.The skilled artisan will also recognize that different independenttransformation events will result in different levels and patterns ofexpression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al.,Mol. Gen. Genetics 218:78-86 (1989)), and thus that multiple events mustbe screened in order to obtain lines displaying the desired expressionlevel and pattern. Such screening may be accomplished by Southernanalysis of DNA, Northern analysis of mRNA expression, immunoblottinganalysis of protein expression, or phenotypic analysis, among others.

The term “expression”, as used herein, refers to the production of afunctional end-product (e.g., a mRNA or a protein [either precursor ormature]).

The term “introduced” means providing a nucleic acid (e.g., expressionconstruct) or protein into a cell. Introduced includes reference to theincorporation of a nucleic acid into a eukaryotic or prokaryotic cellwhere the nucleic acid may be incorporated into the genome of the cell,and includes reference to the transient provision of a nucleic acid orprotein to the cell. Introduced includes reference to stable ortransient transformation methods, as well as sexually crossing. Thus,“introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct/expression construct) into a cell, means“transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid fragment into aeukaryotic or prokaryotic cell where the nucleic acid fragment may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

“Mature” protein refers to a post-translationally processed polypeptide(i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed). “Precursor” protein refers tothe primary product of translation of mRNA (i.e., with pre- andpropeptides still present). Pre- and propeptides may be but are notlimited to intracellular localization signals.

“Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance. Incontrast, “transient transformation” refers to the transfer of a nucleicacid fragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms.

As used herein, “transgenic” refers to a plant or a cell which compriseswithin its genome a heterologous polynucleotide. Preferably, theheterologous polynucleotide is stably integrated within the genome suchthat the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of an expression construct. Transgenic is used herein to includeany cell, cell line, callus, tissue, plant part or plant, the genotypeof which has been altered by the presence of heterologous nucleic acidincluding those transgenics initially so altered as well as thosecreated by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.“Co-suppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of identical or substantiallysimilar foreign or endogenous genes (U.S. Pat. No. 5,231,020).Co-suppression constructs in plants previously have been designed byfocusing on overexpression of a nucleic acid sequence having homology toan endogenous mRNA, in the sense orientation, which results in thereduction of all RNA having homology to the overexpressed sequence(Vaucheret et al., Plant J. 16:651-659 (1998); Gura, Nature 404:804-808(2000)). The overall efficiency of this phenomenon is low, and theextent of the RNA reduction is widely variable. More recent work hasdescribed the use of “hairpin” structures that incorporate all, or part,of an mRNA encoding sequence in a complementary orientation that resultsin a potential “stem-loop” structure for the expressed RNA (PCTPublication No. WO 99/53050, published Oct. 21, 1999; PCT PublicationNo. WO 02/00904, published Jan. 3, 2002). This increases the frequencyof co-suppression in the recovered transgenic plants. Another variationdescribes the use of plant viral sequences to direct the suppression, or“silencing”, of proximal mRNA encoding sequences (PCT Publication No. WO98/36083, published Aug. 20, 1998). Both of these co-suppressingphenomena have not been elucidated mechanistically, although geneticevidence has begun to unravel this complex situation (Elmayan et al.,Plant Cell 10:1747-1757 (1998)).

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of lipid (Weete, In: Fungal LipidBiochemistry, 2nd Ed., Plenum, 1980). A class of plants identified asoleaginous are commonly referred to as “oilseed” plants. Examples ofoilseed plants include, but are not limited to: soybean (Glycine andSofa sp.), flax (Linum sp.), rapeseed (Brassica sp.), maize, cotton,safflower (Carthamus sp.) and sunflower (Helianthus sp.).

Within oleaginous microorganisms the cellular oil or TAG contentgenerally follows a sigmoid curve, wherein the concentration of lipidincreases until it reaches a maximum at the late logarithmic or earlystationary growth phase and then gradually decreases during the latestationary and death phases (Yongmanitchai and Ward, Appl. Environ.Microbial. 57:419-25 (1991)).

Also described herein are oleaginous microbial organisms produced by themethods described herein. This therefore includes oleaginous bacteria,algae, moss, euglenoids, stramenopiles fungi and yeast, comprising intheir genome a recombinant construct incorporating an isolated nucleicacid of the present invention. Additionally, lipids and oils obtainedfrom these oleaginous organisms, products obtained from the processingof the lipids and oil, use of these lipids and oil in foods, animalfeeds or industrial applications and/or use of the by-products in foodsor animal feeds are also described. Examples of microalgae include, butare not limited to Rhodomonas salina, Crypthecodinium cohnii,Chaetoceros lauderi, Pavlova pinguis, and Emiliania huxleyi. There iscurrently great interest in using oleaginous microalgae to produce oilfor biofuels, or for use as nutraceuticals or cosmetics (Hu et al, 2008,Plant J 54:621-639; Waltz, 2009, Nature Biotechnology 27: 15-18.) Theapproach of overexpressing genes in microalgae to improve oil productionfor biofuels applications is being explored (Waltz, 2009, NatureBiotchnology 27:15-18.)

The term “oleaginous yeast” refers to those microorganisms classified asyeasts that make oil. It is not uncommon for oleaginous microorganismsto accumulate in excess of about 25% of their dry cell weight as oil.Examples of oleaginous yeast include, but are no means limited to, thefollowing genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium,Cryptococcus, Trichosporon and Lipomyces.

The term “plant” refers to whole plants, plant organs, plant tissues,seeds, plant cells, seeds and progeny of the same. Plant cells include,without limitation, cells from seeds, suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen and microspores.

“Progeny” comprises any subsequent generation of a plant.

“Non-transgenic, null segregant soybean seed” refers to a near isogenicplant or seed that lacks the transgene, and/or a parental plant used inthe transformation process to obtain the transgenic event. Nullsegregants can be plants or seed that do not contain the transgenictrait due to normal genetic segregation during propagation of theheterozygous transgenic plants.

A “kernel” is the corn caryopsis, consisting of a mature embryo andendosperm which are products of double fertilization. The term “corn” or““maize”” represents any variety, cultivar, or population of Zea mays L.

“Grain” comprises mature corn kernels produced by commercial growers foron farm use or for sale to customers in both cases for purposes otherthan growing or reproducing the species. The ““seed”” is the mature cornkernel produced for the purpose of propagating the species and for saleto commercial growers. As used herein the terms seeds, kernels, andgrains can be used interchangeably. The “embryo” or also termed “germ”is a young sporophytic plant, before the start of a period of rapidgrowth (seed germination). The embryo (germ) of corn contains the vastmajority of the oil found in the kernel. The structure of embryo incereal grain includes the embryonic axis and the scutellum. The“scutellum” is the single cotyledon of a cereal grain embryo,specialized for absorption of the endosperm. The “aleurone” is aproteinaceous material, usually in the form of small granules, occurringin the outermost cell layer of the endosperm of corn and other grains.

Plant oil is a valuable renewable resource, with annual world productionof 145 million metric tons valued at over 80 billion U.S. dollars(Rupilius and Ahmad, 2007, Eur J Lipid Sci Technol 109:433-439).Discovering ways to increase the oil content of plants is thereforedesired. Previous transgenic studies showed that diacylglycerolacyltransferase (DGAT) has a role in controlling oil production inplants.

Ectopic expression of an Arabidopsis type 1 DGAT gene in Arabidopsisincreased seed oil content (Jako et al., 2001, Plant Physiology 126:861-874). Likewise, ectopic expression of maize type I DGAT alleles inmaize increased kernel and embryo oil content (Zheng et al., 2008,Nature Genetics 40:367-372). Increased oil in Brassica napus wasobserved as a result of ectopic expression of Arabidopisis and Brassicanapus type I DGAT genes (Weselake et al., 2008, J Exper Bot 59:3543-3549), or a nasturtium type I DGAT gene (Xu et al, 2008, PlantBiotechnology J 6:799-818). Non-higher plant DGATs have also been used,the type 2 DGAT from the fungus Umbelopsis ramanniana increased oilcontent when expressed in soybean (Lardizabal et al., 2008, PlantPhysiol 148: 89-96).

Discovery of DGAT enzymes with higher activity or with better kinetic orregulatory properties may lead to still greater increases in plant oilcontent than achieved previously. Extremely high oil plant tissues maybe good sources of DGAT genes that encode enzymes with favorableproperties.

“Tree nuts” and edible nuts from some trees and shrubs have very highoil contents in comparison with important oil crops of the world(Gunstone et al., 1994, The Lipid Handbook, 2^(nd) Edition, Chapman andHall, 2-6 Boundary Row, London SE1 8HN, UK, page 112). For example, thenuts of hazelnut and hickory have 62% and 70% oil, respectively. Inaddition to high oil contents, some of the edible nuts also have oilwith a high proportion of oleic acid. For example, the oil of hazelnutand hickory contains 76% and 52% oleic acid, respectively (Gunstone etal., 1994, The Lipid Handbook, 2^(nd) Edition, Chapman and Hall, 2-6Boundary Row, London SE1 8HN, UK, page 112).

The DGAT genes from these species may therefore be especially effectivein high oleic crops that contain a high proportion of oleoyl-CoA anddi-oleoyl diacylglycerol as substrates during the oil formation periodof development. It is therefore of interest to express the DGAT genesobtained from high oil and high oleic tissues in high oleic cropscontaining reduced FAD2 (delta-12 fatty acid desaturase) activity,achieved either through plant breeding or transgenically.

As is discussed in the Examples below a representative of the hazelnutgenus Corylus, a DGAT cDNA from American hazelnut (Corylus americana)was isolated. The hazelnut genus Corylus also includes many otherclosely related species that would be expected to have DGATs withsimilar properties and high sequence identity, including the species forcommon hazelnut (avellana), beaked hazelnut (cornuta), Filbert (maxima),and Turkish hazelnut (colurna), among numerous others.

As is discussed in the Examples below a representative of thehickory/pecan genus Carya, a DGAT cDNAs from shagbark hickory (Caryaovata) was isolated. The hickory/pecan genus Carya also includes manyother closely related species that would be expected to have DGATs withsimilar properties and high sequence identity, including the species forpecan (illinoinensis), shellbark hickory (laciniosa), mockernut hickory(tomentosa), pignut hickory (glabra), bitternut hickory (cordiformis),Chinese hickory (cathayensis), and Vietnamese hickory (tonkinensis),among numerous others.

The method disclosed here to obtain and test DGAT cDNAs from Americanhazelnut and shagbark hickory could also be used to obtain and test DGATcDNAs from these other species of the Corylus or Carya genera, or fromother edible nuts described in (Gunstone et al., 1994, The LipidHandbook, 2^(nd) Edition, Chapman and Hall, 2-6 Boundary Row, London SE18HN, UK, page 112), such as pistachio (57% oil, 69% oleic) for example.

In addition to obtaining DGAT genes with desirable properties from novelsources like tree nuts, protein engineering approaches could also betaken to improve kinetic, regulatory, or other properties of DGAT. ThisDGAT engineering approach has not been thoroughly explored to date.Several amino acid substitutions were made in the nasturtium DGAT, butonly one of these substitutions, a serine to alanine change at position197, resulted in increased specific activity (Xu et al, 2008, PlantBiotechnology J 6:799-818). This position corresponds to serine 216 inthe hazelnut DGAT sequence of the present invention. In another study,multiple random mutations were made in Brassica napus DGAT, and somemutated enzymes increased oil content in yeast (Siloto et al., 2009,Plant Physiol Biochem 47:456-461). However, the identity of themutations was not reported, and no demonstrations of increased oil in aplant tissue as a result of multiple simultaneous amino acidsubstitutions in DGAT have been reported prior to the present studydescribed in the Examples section.

The present invention concerns a transgenic soybean seed havingincreased total fatty acid content of at least 10% when compared to thetotal fatty acid content of a null segregant soybean seed. It isunderstood that any measurable increase in the total fatty acid contentof a transgenic versus a null segregant would be useful. Such increasesin the total fatty acid content would include, but are not limited to,at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, or 20%.

A transgenic oilseed of the invention can comprise a recombinantconstruct having at least one DGAT sequence. This DGAT sequence can beselected from the group consisting of DGAT1, DGAT2 and DGAT1 incombination with DGAT2. Furthermore, at least one DGAT sequence can befrom a tree nut or a shuffled DGAT. Examples of suitable DGAT sequencesthat can be used to practice the invention are discussed in the Examplesbelow. There can be mentioned SEQ ID NOs: 7, 9, 11, 51, 53, 137, 141,143, 147, 156, 160, 163, 165, 167, 169, 184, 186, 188, 190, 192, and194, in the present invention. Those skilled in the art will appreciatethat the instant invention includes, but is not limited to, the DGATsequences disclosed herein.

Such a recombinant construct promoter would comprise differentcomponents such as a promoter which is a DNA sequence that directscellular machinery of a plant to produce RNA from the contiguous codingsequence downstream (3′) of the promoter. The promoter region influencesthe rate, developmental stage, and cell type in which the RNA transcriptof the gene is made. The RNA transcript is processed to produce mRNAwhich serves as a template for translation of the RNA sequence into theamino acid sequence of the encoded polypeptide. The 5′ non-translatedleader sequence is a region of the mRNA upstream of the protein codingregion that may play a role in initiation and translation of the mRNA.The 3′ transcription termination/polyadenylation signal is anon-translated region downstream of the protein coding region thatfunctions in the plant cell to cause termination of the RNA transcriptand the addition of polyadenylate nucleotides to the 3′ end of the RNA.

The origin of the promoter chosen to drive expression of the DGAT codingsequence is not important as long as it has sufficient transcriptionalactivity to accomplish the invention by expressing translatable mRNA forthe desired nucleic acid fragments in the desired host tissue at theright time. Either heterologous or non-heterologous (i.e., endogenous)promoters can be used to practice the invention. For example, suitablepromoters include, but are not limited to: the alpha prime subunit ofbeta conglycinin promoter, the Kunitz trypsin inhibitor 3 promoter, theannexin promoter, the glycinin Gy1 promoter, the beta subunit of betaconglycinin promoter, the P34/Gly Bd m 30K promoter, the albuminpromoter, the Leg A1 promoter and the Leg A2 promoter.

The annexin, or P34, promoter is described in PCT Publication No. WO2004/071178 (published Aug. 26, 2004). The level of activity of theannexin promoter is comparable to that of many known strong promoters,such as: (1) the CaMV 35S promoter (Atanassova et al., Plant Mol. Biol.37:275-285 (1998); Battraw and Hall, Plant Mol. Biol. 15:527-538 (1990);Holtorf et al., Plant Mol. Biol. 29:637-646 (1995); Jefferson et al.,EMBO J. 6:3901-3907 (1987); Wilmink et al., Plant Mol. Biol. 28:949-955(1995)); (2) the Arabidopsis oleosin promoters (Plant et al., Plant Mol.Biol. 25:193-205 (1994); Li, Texas A&M University Ph.D. dissertation,pp. 107-128 (1997)); (3) the Arabidopsis ubiquitin extension proteinpromoters (Callis et al., J Biol. Chem. 265(21):12486-93 (1990)); (4) atomato ubiquitin gene promoter (Rollfinke et al., Gene. 211(2):267-76(1998)); (5) a soybean heat shock protein promoter (Schoffl et al., MolGen Genet. 217(2-3):246-53 (1989)); and, (6) a maize H3 histone genepromoter (Atanassova et al., Plant Mol Biol. 37(2):275-85 (1989)).

Another useful feature of the annexin promoter is its expression profilein developing seeds. The annexin promoter is most active in developingseeds at early stages (before 10 days after pollination) and is largelyquiescent in later stages. The expression profile of the annexinpromoter is different from that of many seed-specific promoters, e.g.,seed storage protein promoters, which often provide highest activity inlater stages of development (Chen et al., Dev. Genet. 10:112-122 (1989);Ellerstrom et al., Plant Mol. Biol. 32:1019-1027 (1996); Keddie et al.,Plant Mol. Biol. 24:327-340 (1994); Plant et al., (supra); Li, (supra)).The annexin promoter has a more conventional expression profile butremains distinct from other known seed specific promoters. Thus, theannexin promoter will be a very attractive candidate whenoverexpression, or suppression, of a gene in embryos is desired at anearly developing stage. For example, it may be desirable to overexpressa gene regulating early embryo development or a gene involved in themetabolism prior to seed maturation.

Following identification of an appropriate promoter suitable forexpression of a specific DGAT-coding sequence, the promoter is thenoperably linked in a sense orientation using conventional means wellknown to those skilled in the art.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.et al., In Molecular Cloning: A Laboratory Manual; 2nd ed.; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, N.Y., 1989 (hereinafter“Sambrook et al., 1989”) or Ausubel, F. M., Brent, R., Kingston, R. E.,Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K., Eds.; InCurrent Protocols in Molecular Biology; John Wiley and Sons: New York,1990 (hereinafter “Ausubel et al., 1990”).

In another aspect, this invention concerns a method method forincreasing the total fatty acid content of a soybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cells) of step (a) having anincreased total fatty acid content of at least 10% when compared to thetotal fatty acid content of a null segregant soybean seed.

Once the recombinant construct has been made, it may then be introducedinto a plant cell of choice by methods well known to those of ordinaryskill in the art (e.g., transfection, transformation andelectroporation). Oilseed plant cells are the preferred plant cells. Thetransformed plant cell is then cultured and regenerated under suitableconditions permitting selection of those transformed soybean cell(s)having an increased total fatty acid content of at least 10% whencompared to the total fatty acid content of a null segregant soybeanseed.

Such recombinant constructs may be introduced into one plant cell; or,alternatively, each construct may be introduced into separate plantcells.

Expression in a plant cell may be accomplished in a transient or stablefashion as is described above.

Also within the scope of this invention are seeds or plant partsobtained from such transformed plants.

Plant parts include differentiated and undifferentiated tissuesincluding, but not limited to the following: roots, stems, shoots,leaves, pollen, seeds, tumor tissue and various forms of cells andculture (e.g., single cells, protoplasts, embryos and callus tissue).The plant tissue may be in plant or in a plant organ, tissue or cellculture.

The term “plant organ” refers to plant tissue or a group of tissues thatconstitute a morphologically and functionally distinct part of a plant.The term “genome” refers to the following: (1) the entire complement ofgenetic material (genes and non-coding sequences) that is present ineach cell of an organism, or virus or organelle; and/or (2) a completeset of chromosomes inherited as a (haploid) unit from one parent.

Methods for transforming dicots (primarily by use of Agrobacteriumtumefaciens) and obtaining transgenic plants have been published, amongothers, for: cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135);soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011); Brassica(U.S. Pat. No. 5,463,174); peanut (Cheng et al. Plant Cell Rep.15:653-657 (1996); McKently et al. Plant Cell Rep. 14:699-703 (1995));papaya (Ling, K. et al. Bio/technology 9:752-758 (1991)); and pea (Grantet al. Plant Cell Rep. 15:254-258 (1995)). For a review of othercommonly used methods of plant transformation see Newell, C. A. (Mol.Biotechnol. 16:53-65 (2000)). One of these methods of transformationuses Agrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F.Microbiol. Sci. 4:24-28 (1987)). Transformation of soybeans using directdelivery of DNA has been published using PEG fusion (PCT Publication No.WO 92/17598), electroporation (Chowrira, G. M. et al., Mol. Biotechnol.3:17-23 (1995); Christou, P. et al., Proc. Natl. Acad. Sci. U.S.A.84:3962-3966 (1987)), microinjection and particle bombardement (McCabe,D. E. et. al., Bio/Technology 6:923 (1988); Christou et al., PlantPhysiol. 87:671-674 (1988)).

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated. The regeneration, development and cultivation of plantsfrom single plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, In: Methodsfor Plant Molecular Biology, (Eds.), Academic: San Diego, Calif.(1988)). This regeneration and growth process typically includes thesteps of selection of transformed cells and culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of thepresent invention containing a desired polypeptide is cultivated usingmethods well known to one skilled in the art.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for: the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.); thegeneration of recombinant DNA fragments and recombinant expressionconstructs; and, the screening and isolating of clones. See, forexample: Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor: NY (1989); Maliga et al., Methods in Plant MolecularBiology, Cold Spring Harbor: NY (1995); Birren et al., Genome Analysis:Detecting Genes, Vol. 1, Cold Spring Harbor: NY (1998); Birren et al.,Genome Analysis: Analyzing DNA, Vol. 2, Cold Spring Harbor: NY (1998);Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer: NY(1997).

Examples of oilseed plants include, but are not limited to: soybean,Brassica species, sunflower, maize, cotton, flax and safflower.

In another aspect, this invention concerns a a transgenic corn kernelhaving increased total fatty acid content of at least 10% when comparedto the total fatty acid content of a null segregant corn kernel. Such atransgenic corn kernel can comprise a recombinant construct having atleast one DGAT sequence. This DGAT sequence can be selected from thegroup consisting of DGAT1, DGAT2, or DGAT1 in combination with DGAT2.

In still another aspect, the present invention concerns a method forincreasing the total fatty acid content of a corn kernel comprising:

(a) transforming at least one corn kernel with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed corn kernel(s) of step (a) having anincreased total fatty acid content of at least 10% when compared to thetotal fatty acid content of a null segregant corn kernel.

The present invention also concerns a transgenic soybean seed havingincreased total fatty acid content of at least 10% and an increasedoleic acid content of at least 25% when compared to the total fatty acidcontent and oleic acid content of a null segregant soybean seed. And thepresent invention further concerns a transgenic soybean having increasedtotal fatty acid content of at least 10% and at least any one of i) anincreased oleic acid content of at least 25%; ii) a decreased linolenicacid content of at least 25%; iii) a decreased linoleic acid content ofat least 4%; iv) a decreased palmitic acid content of at least 8%; andv) an increased stearic acid content of at lease 14% when compared tothe total fatty acid content and oleic, linolenic acid, linoleic acid,palmitic acid or stearic acid, respectively, content of a null segregantsoybean seed.

In still a further aspect, the present invention also concerns a methodfor increasing the total fatty acid content and oleic acid content of asoybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and an increasedoleic acid content of at least 25% when compared to the total fatty acidcontent and oleic acid content of a null segregant soybean seed.

In still yet a further aspect, the present invention concerns a methodfor increasing the total fatty acid content and decreasing linolenicacid content of a soybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and a decreasedlinolenic acid content of at least 25% when compared to the total fattyacid content and oleic acid content of a null segregant soybean seed.

Yet again in a further aspect, the present invention concerns a methodfor increasing the total fatty acid content and decreasing linoleic acidcontent of a soybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and a decreasedlinoleic acid content of at least 4% when compared to the total fattyacid content and oleic acid content of a null segregant soybean seed.

Again in a further aspect, the present invention concerns a method forincreasing the total fatty acid content and decreased palmitic acidcontent of a soybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and a decreasedpalmitic acid content of at least 8% when compared to the total fattyacid content and oleic acid content of a null segregant soybean seed.

In yet another aspect, the present invention concerns a method forincreasing the total fatty acid content and stearic acid content of asoybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 10% and an increasedstearic acid content of at least 14% when compared to the total fattyacid content and oleic acid content of a null segregant soybean seed.

As was discussed above, any of the transgenic oilseeds discussed hereincan comprise a recombinant construct having at least one DGAT sequence.This DGAT sequence can be selected from the group consisting of DGAT1,DGAT2, or DGAT1 in combination with DGAT2. Furthermore, at least oneDGAT sequence can be a tree nut sequence, or a shuffled DGAT sequence.

Transformation of monocotyledons using electroporation, particlebombardment, and Agrobacterium have been reported. Transformation andplant regeneration have been achieved in asparagus (Bytebier et al.,Proc. Natl. Acad. Sci. (USA) 84:5354, (1987)); barley (Wan and Lemaux,Plant Physiol 104:37 (1994)); Zea mays (Rhodes et al., Science 240:204(1988), Gordon-Kamm et al., Plant Cell 2:603-618 (1990), Fromm et al.,Bio/Technology 8:833 (1990), Koziel et al., Bio/Technology 11: 194,(1993), Armstrong et al., Crop Science 35:550-557 (1995)); oat (Somerset al., Bio/Technology 10: 15 89 (1992)); orchard grass (Horn et al.,Plant Cell Rep. 7:469 (1988)); rice (Toriyama et al., TheorAppl. Genet.205:34, (1986); Part et al., Plant Mol. Biol. 32:1135-1148, (1996);Abedinia et al., Aust. J. Plant Physiol. 24:133-141 (1997); Zhang andWu, Theor. Appl. Genet. 76:835 (1988); Zhang et al. Plant Cell Rep.7:379, (1988); Battraw and Hall, Plant Sci. 86:191-202 (1992); Christouet al., Bio/Technology 9:957 (1991)); rye (De la Pena et al., Nature325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409 (1992));tall fescue (Wang et al., Bio/Technology 10:691 (1992)), and wheat(Vasil et al., Bio/Technology 10:667 (1992); U.S. Pat. No. 5,631,152).

Assays for gene expression based on the transient expression of clonednucleic acid constructs have been developed by introducing the nucleicacid molecules into plant cells by polyethylene glycol treatment,electroporation, or particle bombardment (Marcotte et al., Nature335:454-457 (1988); Marcotte et al., Plant Cell 1:523-532 (1989);McCarty et al., Cell 66:895-905 (1991); Hattori et al., Genes Dev.6:609-618 (1992); Goff et al., EMBO J. 9:2517-2522 (1990)).

Transient expression systems may be used to functionally dissect geneconstructs (see generally, Maliga et al., Methods in Plant MolecularBiology, Cold Spring Harbor Press (1995)). It is understood that any ofthe nucleic acid molecules of the present invention can be introducedinto a plant cell in a permanent or transient manner in combination withother genetic elements such as vectors, promoters, enhancers etc.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant organisms and the screening and isolating ofclones, (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989); Maliga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995);Birren et al., Genome Analysis: Detecting Genes, 1, Cold Spring Harbor,N.Y. (1998); Birren et al., Genome Analysis: Analyzing DNA, 2, ColdSpring Harbor, N.Y. (1998); Plant Molecular Biology: A LaboratoryManual, eds. Clark, Springer, New York (1997)).

The transgenic soybean seeds of the invention can be processed to yieldsoy oil, soy products and/or soy by-products.

“Soy products” can include, but are not limited to, those items listedin Table 1A.

TABLE 1A Soy Protein Products Derived from Soybean Seeds^(a) WholeSoybean Products Roasted Soybeans Baked Soybeans Soy Sprouts Soy MilkSpecialty Soy Foods/Ingredients Soy Milk Tofu Tempeh Miso Soy SauceHydrolyzed Vegetable Protein Whipping Protein Processed Soy ProteinProducts Full Fat and Defatted Flours Soy Grits Soy Hypocotyls SoybeanMeal Soy Milk Soy Protein Isolates Soy Protein Concentrates Textured SoyProteins Textured Flours and Concentrates Textured Concentrates TexturedIsolates ^(a)See Soy Protein Products: Characteristics, NutritionalAspects and Utilization (1987). Soy Protein Council.

“Processing” refers to any physical and chemical methods used to obtainthe products listed in Table 1A and includes, but is not limited to,heat conditioning, flaking and grinding, extrusion, solvent extraction,or aqueous soaking and extraction of whole or partial seeds.Furthermore, “processing” includes the methods used to concentrate andisolate soy protein from whole or partial seeds, as well as the varioustraditional Oriental methods in preparing fermented soy food products.Trading Standards and Specifications have been established for many ofthese products (see National Oilseed Processors Association Yearbook andTrading Rules 1991-1992). Products referred to as being “high protein”or “low protein” are those as described by these StandardSpecifications. “NSI” refers to the Nitrogen Solubility Index as definedby the American Oil Chemists' Society Method Ac4 41. “KOH NitrogenSolubility” is an indicator of soybean meal quality and refers to theamount of nitrogen soluble in 0.036 M KOH under the conditions asdescribed by Araba and Dale [(1990) Poult. Sci. 69:76-83]. “White”flakes refer to flaked, dehulled cotyledons that have been defatted andtreated with controlled moist heat to have an NSI of about 85 to 90.This term can also refer to a flour with a similar NSI that has beenground to pass through a No. 100 U.S. Standard Screen size. “Cooked”refers to a soy protein product, typically a flour, with an NSI of about20 to 60. “Toasted” refers to a soy protein product, typically a flour,with an NSI below 20. “Grits” refer to defatted, dehulled cotyledonshaving a U.S. Standard screen size of between No. 10 and 80. “SoyProtein Concentrates” refer to those products produced from dehulled,defatted soybeans by three basic processes: acid leaching (at about pH4.5), extraction with alcohol (about 55-80%), and denaturing the proteinwith moist heat prior to extraction with water. Conditions typicallyused to prepare soy protein concentrates have been described by Pass[(1975) U.S. Pat. No. 3,897,574; Campbell et al., (1985) in New ProteinFoods, ed. by Altschul and Wilcke, Academic Press, Vol. 5, Chapter 10,Seed Storage Proteins, pp 302-338]. “Extrusion” refers to processeswhereby material (grits, flour or concentrate) is passed through ajacketed auger using high pressures and temperatures as a means ofaltering the texture of the material. “Texturing” and “structuring”refer to extrusion processes used to modify the physical characteristicsof the material. The characteristics of these processes, includingthermoplastic extrusion, have been described previously [Atkinson (1970)U.S. Pat. No. 3,488,770, Horan (1985) In New Protein Foods, ed. byAltschul and Wilcke, Academic Press, Vol. 1A, Chapter 8, pp 367414].Moreover, conditions used during extrusion processing of complexfoodstuff mixtures that include soy protein products have been describedpreviously [Rokey (1983) Feed Manufacturing Technology III, 222-237;McCulloch, U.S. Pat. No. 4,454,804].

TABLE 1B Generalized Steps for Soybean Oil and Byproduct ProductionProcess Impurities Removed and/or Step Process By-Products Obtained # 1soybean seed # 2 oil extraction meal # 3 Degumming lecithin # 4 alkalior physical gums, free fatty acids, refining pigments # 5 water washingsoap # 6 Bleaching color, soap, metal # 7 (hydrogenation) # 8(winterization) stearine # 9 Deodorization free fatty acids,tocopherols, sterols, volatiles # 10  oil products

More specifically, soybean seeds are cleaned, tempered, dehulled, andflaked, thereby increasing the efficiency of oil extraction. Oilextraction is usually accomplished by solvent (e.g., hexane) extractionbut can also be achieved by a combination of physical pressure and/orsolvent extraction. The resulting oil is called crude oil. The crude oilmay be degummed by hydrating phospholipids and other polar and neutrallipid complexes that facilitate their separation from the nonhydrating,triglyceride fraction (soybean oil). The resulting lecithin gums may befurther processed to make commercially important lecithin products usedin a variety of food and industrial products as emulsification andrelease (i.e., antisticking) agents. Degummed oil may be further refinedfor the removal of impurities (primarily free fatty acids, pigments andresidual gums). Refining is accomplished by the addition of a causticagent that reacts with free fatty acid to form soap and hydratesphosphatides and proteins in the crude oil. Water is used to wash outtraces of soap formed during refining. The soapstock byproduct may beused directly in animal feeds or acidulated to recover the free fattyacids. Color is removed through adsorption with a bleaching earth thatremoves most of the chlorophyll and carotenoid compounds. The refinedoil can be hydrogenated, thereby resulting in fats with various meltingproperties and textures. Winterization (fractionation) may be used toremove stearin from the hydrogenated oil through crystallization undercarefully controlled cooling conditions. Deodorization (principally viasteam distillation under vacuum) is the last step and is designed toremove compounds which impart odor or flavor to the oil. Other valuablebyproducts such as tocopherols and sterols may be removed during thedeodorization process. Deodorized distillate containing these byproductsmay be sold for production of natural vitamin E and other high-valuepharmaceutical products. Refined, bleached, (hydrogenated, fractionated)and deodorized oils and fats may be packaged and sold directly orfurther processed into more specialized products. A more detailedreference to soybean seed processing, soybean oil production, andbyproduct utilization can be found in Erickson, Practical Handbook ofSoybean Processing and Utilization, The American Oil Chemists' Societyand United Soybean Board (1995). Soybean oil is liquid at roomtemperature because it is relatively low in saturated fatty acids whencompared with oils such as coconut, palm, palm kernel, and cocoa butter.

Plant and microbial oils containing PUFAs that have been refined and/orpurified can be hydrogenated, thereby resulting in fats with variousmelting properties and textures. Many processed fats (including spreads,confectionary fats, hard butters, margarines, baking shortenings, etc.)require varying degrees of solidity at room temperature and can only beproduced through alteration of the source oil's physical properties.This is most commonly achieved through catalytic hydrogenation.

Hydrogenation is a chemical reaction in which hydrogen is added to theunsaturated fatty acid double bonds with the aid of a catalyst such asnickel. For example, high oleic soybean oil contains unsaturated oleic,linoleic, and linolenic fatty acids, and each of these can behydrogenated. Hydrogenation has two primary effects. First, theoxidative stability of the oil is increased as a result of the reductionof the unsaturated fatty acid content. Second, the physical propertiesof the oil are changed because the fatty acid modifications increase themelting point resulting in a semi-liquid or solid fat at roomtemperature.

There are many variables which affect the hydrogenation reaction, whichin turn alter the composition of the final product. Operating conditionsincluding pressure, temperature, catalyst type and concentration,agitation, and reactor design are among the more important parametersthat can be controlled. Selective hydrogenation conditions can be usedto hydrogenate the more unsaturated fatty acids in preference to theless unsaturated ones. Very light or brush hydrogenation is oftenemployed to increase stability of liquid oils. Further hydrogenationconverts a liquid oil to a physically solid fat. The degree ofhydrogenation depends on the desired performance and meltingcharacteristics designed for the particular end product. Liquidshortenings (used in the manufacture of baking products, solid fats andshortenings used for commercial frying and roasting operations) and basestocks for margarine manufacture are among the myriad of possible oiland fat products achieved through hydrogenation. A more detaileddescription of hydrogenation and hydrogenated products can be found inPatterson, H. B. W., Hydrogenation of Fats and Oils: Theory andPractice. The American Oil Chemists' Society (1994).

Hydrogenated oils have become somewhat controversial due to the presenceof trans-fatty acid isomers that result from the hydrogenation process.Ingestion of large amounts of trans-isomers has been linked withdetrimental health effects including increased ratios of low density tohigh density lipoproteins in the blood plasma and increased risk ofcoronary heart disease.

In another aspect the present invention concerns an isolatedpolynucleotide comprising:

(a) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity wherein the polypeptide has at least 80% aminoacid identity, based on the Clustal V method of alignment, when comparedto an amino acid sequence as set forth in SEQ ID NOs:8, 10, or 12;

(b) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity, wherein the nucleotide sequence has at least80% sequence identity, based on the BLASTN method of alignment, whencompared to a nucleotide sequence as set forth in SEQ ID NO: 7, 9, or11:

(c) a nucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity, wherein the nucleotide sequence hybridizesunder stringent conditions to a nucleotide sequence as set forth in SEQID NO: 7, 9, or 11; or

(d) a complement of the nucleotide sequence of (a), (b) or (c), whereinthe complement and the nucleotide sequence consist of the same number ofnucleotides and are 100% complementary.

The isolated polynucleotide may be obtained from one or more ediblenuts, such as, but not limited to, hazelnut, hickory, pistachio, andpecan. The isolated polynucleotide may also be part of a recombinant DNAconstruct comprising at least one regulatory sequence. This recombinantconstruct may also be comprised in a cell. This cell may be from anoilseed plant. Suitable oilseed plants include, but are not limited to,soybean, corn, canola, sunflower, flax, cotton, and safflower.

In a further aspect the present invention concerns a method forincreasing the total fatty acid content of an oilseed comprising:

(a) transforming at least one oilseed cell with the above mentionedrecombinant construct;

(b) selecting the transformed oilseed cell(s) of step (a) having anincreased total fatty acid content when compared to the total fatty acidcontent of a null segregant oilseed.

Polynucleotide sequences produced by diversity generation methods orrecursive sequence recombination (“RSR”) methods (e.g., DNA shuffling),which can be accomplished in vitro, in vivo, in silica, or a combinationthereof are a feature of the invention. A diversification method caninclude recursively recombining one or more nucleotide sequences of theinvention as described below with one or more additional nucleotides.The recombining steps are optionally performed in vivo, ex vivo, insilica or in vitro. This diversity generation or recursive sequencerecombination produces at least one library of recombinant modified DGATpolynucleotides. Polypeptides encoded by members of this library areincluded in the invention. These polypeptides can be referred to, butare not limited to, terms such as “shuffled DGATs”, modified Type Idiacylglycerol acyltransferase”, “modified DGATs”, or “DGAT sequencescontaining amino acid substitutions resulting in oil increases”.

DGATs of the present invention can be readily modified using methodsthat are well known in the art to improve or alter DGAT activity. Avariety of diversity generating protocols are available and described inthe art. The procedures can be used separately, and/or in combination toproduce one or more variants of a nucleic acid or set of nucleic acids,as well as variants of encoded proteins. Individually and collectively,these procedures provide robust, widely applicable ways of generatingdiversified nucleic acids and sets of nucleic acids (including, nucleicacid libraries) which are useful for the engineering or rapid evolutionof nucleic acids, proteins, pathways, cells and/or organisms with newand/or improved characteristics.

While distinctions and classifications are made in the course of theensuing discussion for clarity; it will be appreciated that thetechniques are often not mutually exclusive. Indeed, the various methodscan be used singly or in combination, in parallel or in series, toaccess diverse sequence variants.

The result of any of the diversity generating procedures describedherein can be the generation of one or more nucleic acids, which can beselected or screened for nucleic acids that encode proteins with orwhich confer desirable properties. Following diversification by one ormore of the methods herein, or otherwise available to one of skill, anynucleic acids that are produced can be selected for a desired activityor property, e.g. DGAT activity. A variety of related (or evenunrelated) properties can be evaluated, in serial or in parallel, at thediscretion of the practitioner.

Descriptions of a variety of diversity generating procedures, includingmultigene shuffling and methods for generating modified nucleic acidsequences encoding multiple enzymatic domains, are found in, e.g., thefollowing publications and the references cited therein: Soong, N. etal. (2000) “Molecular breeding of viruses” Nat Genet. 25(4):436-39;Stemmer, et al. (1999) “Molecular breeding of viruses for targeting andother clinical properties” Tumor Targeting 4:1-4; Ness et al. (1999)“DNA Shuffling of subgenomic sequences of subtilisin” NatureBiotechnology 17:893-896; Chang et al. (1999) “Evolution of a cytokineusing DNA family shuffling” Nature Biotechnology 17:793-797; Minshulland Stemmer (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians et al. (1999)“Directed evolution of thymidine kinase for AZT phosphorylation usingDNA family shuffling” Nature Biotechnology 17:259-264; Crameri et al.(1998) “DNA shuffling of a family of genes from diverse speciesaccelerates directed evolution” Nature 391:288-291; Crameri et al.(1997) “Molecular evolution of an arsenate detoxification pathway by DNAshuffling,” Nature Biotechnology 15:436-438; Zhang et al. (1997)“Directed evolution of an effective fucosidase from a galactosidase byDNA shuffling and screening” Proc. Natl. Acad. Sci. USA 94:4504-4509;Patten et al. (1997) “Applications of DNA Shuffling to Pharmaceuticalsand Vaccines” Current Opinion in Biotechnology 8:724-733; Crameri e al.(1996) “Construction and evolution of antibody-phage libraries by DNAshuffling” Nature Medicine 2:100-103; Crameri et al. (1996) “Improvedgreen fluorescent protein by molecular evolution using DNA shuffling”Nature Biotechnology 14:315-319; Gates et al. (1996) “Affinity selectiveisolation of ligands from peptide libraries through display on a lacrepressor ‘headpiece dimer’” Journal of Molecular Biology 255:373-386;Stemmer (1996) “Sexual PCR and Assembly PCR” In: The Encyclopedia ofMolecular Biology. VCH Publishers, New York, pp. 447-457; Crameri andStemmer (1995) “Combinatorial multiple cassette mutagenesis creates allthe permutations of mutant and wildtype cassettes” BioTechniques18:194-195; Stemmer et al., (1995) “Single-step assembly of a gene andentire plasmid from large numbers of oligodeoxy-ribonucleotides” Gene,164:49-53; Stemmer (1995) “The Evolution of Molecular Computation”Science 270: 1510; Stemmer (1995) “Searching Sequence Space”Bio/Technology 13:549-553; Stemmer (1994) “Rapid evolution of a proteinin vitro by DNA shuffling” Nature 370:389-391; and Stemmer (1994) “DNAshuffling by random fragmentation and reassembly: In vitro recombinationfor molecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Additional details regarding various diversity generating methods can befound in, e.g., the following U.S. patents, PCT publications, and EPOpublications: U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252,and 5,837,458; WO 95/22625; WO 96/33207; WO 97/20078; WO 97/35966; WO99/41402; WO 99/41383; WO 99/41369; WO 99/41368; EP 752008; EP 0932670;WO 99/23107; WO 99/21979; WO 98/31837; WO 98/27230; WO 98/13487; WO00/00632; WO 00/09679; WO 98/42832; WO 99/29902; WO 98/41653; WO98/41622; WO 98/42727; WO 00/18906; WO 00/04190; WO 00/42561; WO00/42559; WO 00/42560; WO 01/23401; and WO 01/64864.

Additional details regarding various diversity generating methods can befound in, e.g., U.S. patent application Ser. Nos. 09/407,800 and60/186,482; U.S. Pat. Nos. 6,379,964, 6,376,246, 6,436,675, 6,642,426,and 7,024,312; WO 00/42561; WO 00/42560; and WO 00/42559.

In brief, several different general classes of sequence modificationmethods, such as mutation, recombination, etc. are set forth in thereferences above. Accordingly, the DGAT nucleic acids of the inventioncan be generated from wild type sequences. Moreover, the DGAT nucleicacid sequences of the invention can be modified to create modifiedsequences with the same or different activity.

Synthetic recombination methods can also be used, in whicholigonucleotides corresponding to targets of interest are synthesizedand reassembled in PCR or ligation reactions which includeoligonucleotides which correspond to more than one parental nucleicacid, thereby generating new recombined nucleic acids. Oligonucleotidescan be made by standard nucleotide addition methods, or can be made,e.g., by tri-nucleotide synthetic approaches. Details regarding suchapproaches are found in the references noted above, including, e.g., WO00/42561, WO 01/23401, WO 00/42560, and WO 00/42559.

In silica methods of recombination can be effected in which geneticalgorithms are used in a computer to recombine sequence strings whichcorrespond to homologous (or even non-homologous) nucleic acids. Theresulting recombined sequence strings are optionally converted intonucleic acids by synthesis of nucleic acids which correspond to therecombined sequences, e.g., in concert with oligonucleotide synthesisgene reassembly techniques. This approach can generate random, partiallyrandom or designed variants. Many details regarding in silicarecombination, including the use of genetic algorithms, geneticoperators and the like in computer systems, combined with generation ofcorresponding nucleic acids (and/or proteins), as well as combinationsof designed nucleic acids and/or proteins (e.g., based on cross-oversite selection) as well as designed, pseudo-random or randomrecombination methods are described in WO 00/42560 and WO 00/42559.Extensive details regarding in silica recombination methods are found inthese applications. This methodology is generally applicable to thepresent invention in providing for recombination of nucleic acidsequences and/or gene fusion constructs encoding DGAT proteins in silicaand/or the generation of corresponding nucleic acids or proteins.

Many of the above-described methodologies for generating modifiedpolynucleotides generate a large number of diverse variants of aparental sequence or sequences. In some preferred embodiments of theinvention, the modification technique (e.g., some form of shuffling) isused to generate a library of variants that is then screened for amodified polynucleotide or pool of modified polynucleotides encodingsome desired functional attribute, e.g., improved DGAT activity.Exemplary enzymatic activities that can be screened for includecatalytic rates (conventionally characterized in terms of kineticconstants such as k_(cat) and K_(M)), substrate specificity, andsusceptibility to activation or inhibition by substrate, product orother molecules (e.g., inhibitors or activators).

In another aspect, the present inventionconcerns an isolated nucleicacid fragment, and methods of using said fragment, encoding a modifiedType 1 diacylglycerol acyltransferase polypeptide such that the modifiedType 1 diacylglycerol acyltransferase polypeptide has at least one aminoacid substitution selected from the group consisting of: a non-alanineat a position corresponding to position 12 of SEQ ID NO:12 to alanine, anon-proline at a position corresponding to position 30 of SEQ ID NO:12to proline, a non-alanine at a position corresponding to position 31 ofSEQ ID NO:12 to alanine, a non-serine at a position corresponding toposition 48 of SEQ ID NO:12 to serine, a non-serine at a positioncorresponding to position 49 of SEQ ID NO:12 to serine, a non-aspartateat a position corresponding to position 51 of SEQ ID NO:12 to aspartate,a non-aspartate at a position corresponding to position 52 of SEQ IDNO:12 to aspartate, a non-threonine at a position corresponding toposition 59 of SEQ ID NO:12 to threonine, a non-threonine at a positioncorresponding to position 73 of SEQ ID NO:12 to threonine, anon-asparagine at a position corresponding to position 79 of SEQ IDNO:12 to sparagine, a non-leucine at a position corresponding toposition 118 of SEQ ID NO:12 to leucine, a non-alanine at a positioncorresponding to position 123 of SEQ ID NO:12 to alanine, a non-valineat a position corresponding to position 128 of SEQ ID NO:12 to valine, anon-leucine at a position corresponding to position 139 of SEQ ID NO:12to leucine, a non-isoleucine at a position corresponding to position 155of SEQ ID NO:12 to isoleucine, a non-alanine at a position correspondingto position 181 of SEQ ID NO:12 to alanine, a non-serine at a positioncorresponding to position 184 of SEQ ID NO:12 to serine, a non-valine ata position corresponding to position 197 of SEQ ID NO:12 to valine, anon-valine at a position corresponding to position 198 of SEQ ID NO:12to valine, a non-methionine at a position corresponding to position 205of SEQ ID NO:12 to methionine, a non-threonine at a positioncorresponding to position 211 of SEQ ID NO:12 to threonine, anon-histidine at a position corresponding to position 218 of SEQ IDNO:12 to histidine, a non-valine at a position corresponding to position222 of SEQ ID NO:12 to valine, a non-lysine at a position correspondingto position 241 of SEQ ID NO:12 to lysine, a non-valine at a positioncorresponding to position 247 of SEQ ID NO:12 to valine, a non-valine ata position corresponding to position 251 of SEQ ID NO:12 to valine, anon-serine at a position corresponding to position 256 of SEQ ID NO:12to serine, a non-serine at a position corresponding to position 257 ofSEQ ID NO:12 to serine, a non-phenylalanine at a position correspondingto position 266 of SEQ ID NO:12 to henylalanine, a non-alanine at aposition corresponding to position 267 of SEQ ID NO:12 to alanine, anon-glutamate at a position corresponding to position 281 of SEQ IDNO:12 to glutamate, a non-aspartate at a position corresponding toposition 288 of SEQ ID NO:12 to aspartate, a non-glutamate at a positioncorresponding to position 293 of SEQ ID NO:12 to glutamate, anon-asparagine at a position corresponding to position 294 of SEQ IDNO:12 to asparagine, a non-threonine at a position corresponding toposition 299 of SEQ ID NO:12 to threonine, a non-asparagine at aposition corresponding to position 301 of SEQ ID NO:12 to asparagine, anon-leucine at a position corresponding to position 308 of SEQ ID NO:12to leucine, a non-glycine at a position corresponding to position 327 ofSEQ ID NO:12 to glycine, a non-leucine at a position corresponding toposition 329 of SEQ ID NO:12 to leucine, a non-leucine at a positioncorresponding to position 334 of SEQ ID NO:12 to leucine, a non-valineat a position corresponding to position 337 of SEQ ID NO:12 to valine, anon-valine at a position corresponding to position 338 of SEQ ID NO:12to valine, a non-glutamine at a position corresponding to position 356of SEQ ID NO:12 to glutamine, a non-asparagine at a positioncorresponding to position 363 of SEQ ID NO:12 to asparagine, anon-serine at a position corresponding to position 390 of SEQ ID NO:12to serine, a non-valine at a position corresponding to position 399 ofSEQ ID NO:12 to valine, a non-isoleucine at a position corresponding toposition 436 of SEQ ID NO:12 to isoleucine, a non-alanine at a positioncorresponding to position 451 of SEQ ID NO:12 to alanine, a non-serineat a position corresponding to position 457 of SEQ ID NO:12 to serine, anon-methionine at a position corresponding to position 475 of SEQ IDNO:12 to methionine, a non-phenylalanine at a position corresponding toposition 486 of SEQ ID NO:12 to phenylalanine, a non-isoleucine at aposition corresponding to position 488 of SEQ ID NO:12 to isoleucine, anon-leucine at a position corresponding to position 491 of SEQ ID NO:12to leucine, a non-lysine at a position corresponding to position 502 ofSEQ ID NO:12 to lysine, a non-serine at a position corresponding toposition 514 of SEQ ID NO:12 to serine, a non-valine at a positioncorresponding to position 518 of SEQ ID NO:12 to valine, and anon-valine at a position corresponding to position 531 of SEQ ID NO:12to valine, when compared to the unmodified Type 1 diacylglycerolacyltransferase polypeptide, wherein the position corresponding to aposition of SEQ ID NO:12 is based on an alignment using Clustal V of SEQID NO:12 and the unmodified Type 1 diacylglycerol acyltransferasepolypeptide.

Furthermore, the present invention also concerns an isolated nucleicacid fragment, and methods of using said isolated nucleic acid fragment,encoding a modified Type 1 diacylglycerol acyltransferase polypeptidesuch that the modified Type 1 diacylglycerol acyltransferase polypeptidehas at least one amino acid substitution selected from the groupconsisting of a non-alanine at a position corresponding to position 24of SEQ ID NO:153 to alanine, a non-asparagine at a positioncorresponding to position 58 of SEQ ID NO:153 to asparagine, anon-alanine at a position corresponding to position 146 of SEQ ID NO:153to alanine, a non-methionine at a position corresponding to position 170of SEQ ID NO:153 to methionine, a non-lysine at a position correspondingto position 206 of SEQ ID NO:153 to lysine, a non-valine at a positioncorresponding to position 216 of SEQ ID NO:153 to valine, anon-phenylalanine at a position corresponding to position 231 of SEQ IDNO:153 to phenylalanine, a non-glutamate at a position corresponding toposition 258 of SEQ ID NO:153 to glutamate, a non-threonine at aposition corresponding to position 264 of SEQ ID NO:153 to threonine, anon-leucine at a position corresponding to position 273 of SEQ ID NO:153to leucine, a non-leucine at a position corresponding to position 299 ofSEQ ID NO:153 to leucine, a non-valine at a position corresponding toposition 303 of SEQ ID NO:153 to valine, a non-serine at a positioncorresponding to position 355 of SEQ ID NO:153 to serine, a non-valineat a position corresponding to position 364 of SEQ ID NO:153 to valine,a non-arginine at a position corresponding to position 401 of SEQ IDNO:153 to arginine, a non-serine at a position corresponding to position422 of SEQ ID NO:153 to serine, a non-methionine at a positioncorresponding to position 440 of SEQ ID NO:153 to methionine, anon-lysine at a position corresponding to position 467 of SEQ ID NO:153to lysine, a non-serine at a position corresponding to position 479 ofSEQ ID NO:153 to serine, a non-valine at a position corresponding toposition 483 of SEQ ID NO:153 to valine, when compared to the unmodifiedType 1 diacylglycerol acyltransferase polypeptide, wherein the positioncorresponding to a position of SEQ ID NO:153 is based on an alignmentusing Clustal V of SEQ ID NO:153 and the unmodified Type 1diacylglycerol acyltransferase polypeptide.

It is appreciated the present invention includes plants and progenyincorporating the above mentioned isolated nucleic acid. Other changesin amino acid positions that may contribute to creating a DGATpolypeptide that can results in increased fatty acid levels in plantscan be found in the shuffled hazelnut sequences (SEQ ID NOs:25-150), thealtered maize DGATs (SEQ ID NOs:163-170), and the altered soybean DGATs(SEQ ID NOs:152-161 and 184-195). Any one, any combination, or all ofthese changes are useful for making an altered DGAT that, when expressedin a plant, results in increased oil accumulation. Preferred embodimentsof the invention would include, but are not limited to, at least 1, 2,3, 4, 5, 6, 7, 8. 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aminoacid changes within a DGAT polypeptide, at the positions mentionedabove.

In a final aspect the present invention concerns a fungal cell, oroleaginous microbial organism, comprising a recombinant DNA constructcomprising any isolated nucleic acid fragment encoding anydiacylglycerol acyltransferase of the present invention. Further, thefungal cell can be, but is not limited to, Yarrowia, Candida,Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “mole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s) and “kB” means kilobase(s).

Example 1 Cloning Type 1 DGAT cDNAs from Hickory and Hazelnut

Developing nuts from hickory (Carya ovata), and hazelnut (Corylusamericana), were harvested. The shells were cracked open, and the edibleportion was removed and quickly frozen in liquid nitrogen, followed byfurther storage in a −80° C. freezer until needed. The frozen materialfrom one nut each of hickory and hazelnut were ground separately underliquid nitrogen with a mortar and pestle. Total RNA was isolated fromeach by using a Purescript RNA kit from Gentra Systems (since purchasedby Qiagen), and subsequently poly A RNA was isolated from hickory byusing an mRNA kit from GE Biosciences. Using poly A RNA as template forhickory, and total RNA as template for hazelnut, first strand cDNAsynthesis was done with a SuperScript III kit purchased from Invitrogenusing oligo d(T) as primer and following the manufacturer's protocol.

To obtain partial length hickory and hazelnut DGAT cDNAs, PCR wasperformed with first strand cDNA as template, using primers P21 and P18from conserved regions of known type 1 DGAT genes. The nucleotidesequence of forward primer P21 was 5′-CAAGGAGAGTCCGCTTAGCTC-3′. Thenucleotide sequence of reverse primer P18 was5′-CAGAAAATGAACCAGAAGATCATGTT-3′. The PCR was done using three cycles of94° C. for 30 sec/43° C. for 30 sec/72° C. for 2 min, followed by 35cycles of 94° C. for 30 sec/55° C. for 30 sec/72° C. for 2 min, followedby a final extension of 72° C. for 10 min. PCR products were sequencedto verify that they had sequence homology with known DGAT cDNAs.

The remainder of the DGAT coding regions were obtained with the 5′ RACEand 3′ RACE systems from Invitrogen, using gene specific primersaccording to the manufacturer's protocols. A final PCR was then done toamplify the entire coding region and to include restriction sites tofacilitate cloning. The PCR was done using three cycles of 94° C. for 30sec/43° C. for 30 sec/72° C. for 2 min, followed by 35 cycles of 94° C.for 30 sec/55° C. for 30 sec/72° C. for 2 min, followed by a finalextension of 72° C. for 10 min. Nucleotide sequences of PCR primers usedfor hickory were: forward primer P33,5′-TTTTGGATCCATGGCGATTTCGGATATGCCTG-3′, and reverse primer P34,5′-TTTTCCCGGGTTATTCAGTCTGCCCTTTTCGGTTC-3′. Nucleotide sequences of PCRprimers used for hazelnut were: forward primer P37b,5′-TTTTAGATCTATGGCGATTTCGGATATGCCTGAAAGCACG-3′, and reverse primer P38,5′-TTTTCCCGGGTTATTCAGTCTTCCCTTTACGGTTCATC-3′. The resulting PCR productscontaining the entire coding region were digested by BamH I and Sma Ifor hickory, or Bgl II and Sma I for hazelnut, and ligated into the BamHI and Sma I sites of the yeast expression vector pSZ378 (SEQ ID NO: 22,and FIG. 1). The pSZ378 vector was made by purchasing pRS426 fromStratagene, and adding regions of approximately 1.0 kb promoter and 0.5kb terminator of the S. cerevisiea PGK1 gene that encodes3-phosphoglycerate kinase. This vector may also be used to transform E.coli for routine plasmid DNA preparations, but not for DGAT expression.

Two closely related hickory DGAT cDNAs, and one hazelnut DGAT cDNA wereobtained and named CO-DGAT1a, CO-DGAT1b, and CA-DGAT1, respectively. Thecorresponding nucleotide sequences are SEQ ID NOs: 7, 9, and 11,respectively. The corresponding amino acid sequences are SEQ ID NOs: 8,10, and 12, respectively. The corresponding plasmid names for the DGATgenes (the terms “gene” and “cDNA” are used interchangeably in theseEXAMPLES) following ligation into the yeast expression vector arePHP32238 (SEQ ID NO: 13), PHP 32396 (SEQ ID NO: 14), and PHP32395 (SEQID NO: 15), respectively. The amino acid sequence identities wereanalyzed for the edible nut DGATs and for other type I DGATs fromsoybean (SEQ ID NO: 16, PCT Pub WO 00/32756), Arabidopsis (SEQ ID NO:17, accession # CAB45373), wheat (SEQ ID NO:18, PCT Pub WO 00/32756),and maize (SEQ ID NO:19, accession # EUO39830, presented in NatureGenetics 40:367-372) using the Clustal V sequence alignment program(Table 2). The two hickory DGAT sequences differ by only 3 amino acids.

TABLE 2 DGAT Amino Acid Sequence Identities (%) Hickory Hickory Hazelnut(CO-DGAT1a) (CO-DGAT1b) (CA-DGAT1) Soy Arab. Wheat Maize Hickory 100 9983 73 66 65 66 (CO-DGAT1a) Hickory 100 83 73 66 64 66 (CO-DGAT1b)Hazelnut 100 76 67 64 64 (CA-DGAT1) Soy 100 66 61 63 Arab. 100 59 61Wheat 100 74 Maize 100

Example 2

Expression of Hickory and Hazelnut DGAT cDNAs in Yeast and Determinationof DGAT Activity

A double null Saccharomyces cerevisiae strain with deletions of the DGA1gene that encodes DGAT and the LRO1 gene that encodesphospholipid:diacylglycerol acyltransferase (PDAT) was created as an oildeficient strain suitable for ectopic expression of DGAT cDNAs fromhazelnut and hickory. The double null strain was made by purchasing thestrain deficient in the DGA1 (DGAT) gene from Invitrogen (Clone ID:12501) and then removing the LRO1 (PDAT) gene using homologousrecombination.

Microsomal membrane preparations from yeast cultures transformed withthe hazel and hickory DGAT expression vectors PHP32238, PHP32396, andPHP32395, and with the no DGAT control vector pSZ378, were used for DGATactivity assays. For microsomal membrane preparations, the method ofMilcamps et al, J Biol Chem 280:5370-5377, was followed, with minorchanges. Saccharomyces ceriviseae cultures were grown to earlystationary phase in 100 ml of SC media minus uracil (20 g glucose, 6.7 gDifeo yeast N base w/o amino acids, and 0.77 g—Ura DO supplement/liter).Following harvest, the yeast pellets were resuspended in 4 ml of 20 mMTris-HCl, pH 8, 10 mM MgCl₂, 1 mM EDTA, 5% glycerol, 1 mM DTT, and 0.3 M(NH₄)₂SO₄. Two ml of glass beads (425-600 μm, Sigma catalog #G8772) wereadded, and cells were lysed by vortexing for 5 min. The lysate wascentrifuged for 15 min at 1500 g at 6° C. The supernatant was thencentrifuged at 100,000 g for 1.5 h at 6° C. The microsomal pellet wasresuspended in 500 μl of 100 mM potassium phosphate (pH 7.2) containing10% glycerol, and frozen in liquid nitrogen prior to storage at −80° C.Protein concentrations were determined by the method of Bradford, usingthe Coomassie Plus reagent (Pierce), with bovine serum albumin asstandard.

DGAT assays were done for 1 min at 25° C. with 50 mM potassium phosphatepH 7.2, 10 μM 1-¹⁴C-labelled oleoyl-coenzyme A (Perkin Elmer), and 20 μgof microsomal protein, using endogenous diacylglycerol, in a totalreaction volume of 100 μl. The reaction was started by addition of themicrosomal membranes to the remainder of the reaction components. Theassay was stopped and lipids were extracted with 2 ml ofhexane:isopropanol (3:2) (Hara and Radin, Anal. Biochem. 90:420-426)containing 4 μl of unlabeled triacylglycerol (triolein, Sigma catalog#T7140). Following vortexing for 10 s, the phases were separated with 1ml of 500 mM sodium sulfate and vortexing was again done for 10 s. After10 m, the upper phase was transferred to another tube and dried withnitrogen gas. The lipid was resolubilized in a small volume of hexane(approximately 100 to 150 μl) and applied to K6 silica TLC plates, whichwere developed in 80:20:1 (v/v) hexane:diethylether:acetic acid.Triacylglycerol was visualized and marked by staining in iodine vapor.After the stain faded, the triacylglycerol was scraped, andradioactivity was determined by liquid scintillation counting.

All three DGAT cDNAs were functional (TABLE 3), as indicated by muchgreater activity than observed for the vector control.

TABLE 3 DGAT Activity Following Expression in Yeast DGAT Activity (pmolC14-labeled DGAT cDNA expressed in oleoyl-CoA incorporated into TAGSaccharomyces cerevisiae per min per mg microsomal protein). DGAT/PDATnull Mean ± SD of duplicate assays. Hazelnut (CA-DGAT1) 3622 ± 208Hickory (CO-DGAT1a) 3004 ± 190 Hickory (CO-DGAT1b) 2905 ± 143 Vectorcontrol (no DGAT) 16.7 ± 3.9

Example 3 Expression of CA-DGAT1 in Soybean Somatic Embryos IncreasesOil Content and Oleic Acid Content

A soybean transformation vector KS 394 (SEQ ID NO: 20) was constructedthat included the promoter from the soybean β-conglycinin α40 subunit(Beachy et al., EMBO J. 4:3047-3053) driving expression of the wild typehazel DGAT cDNA CA-DGAT1. A control vector KS352 (SEQ ID NO: 21) thatcontained no DGAT genes was also constructed.

Soybean embryogenic suspension cultures were transformed with intactplasmid DNA of KS 394 or KS 352 by the method of particle gunbombardment (Klein et al., Nature 327:70 (1987)) using a DuPontBiolistic PDS1000/HE instrument (helium retrofit). The tissue cultureand transformation methods are described in more detail as follows:

Culture Conditions:

Soybean embryogenic suspension cultures (cv. Jack) were maintained in 35mL liquid medium SB196 (infra) on a rotary shaker, 150 rpm, 26° C. withcool white fluorescent lights on 16:8 h day/night photoperiod at lightintensity of 60-85 μE/m2/s. Cultures were subcultured every 7 days totwo weeks by inoculating approximately 35 mg of tissue into 35 mL offresh liquid SB196 (the preferred subculture interval is every 7 days).

Soybean Embryogenic Suspension Culture Initiation:

Soybean cultures were initiated twice each month with 5-7 days betweeneach initiation. Pods with immature seeds from available soybean plants45-55 days after planting were picked, removed from their shells andplaced into a sterilized magenta box. The soybean seeds were sterilizedby shaking them for 15 min in a 5% Clorox solution with 1 drop of ivorysoap (i.e., 95 mL of autoclaved distilled water plus 5 mL Clorox and 1drop of soap, mixed well). Seeds were rinsed using 2 1-liter bottles ofsterile distilled water and those less than 4 mm were placed onindividual microscope slides. The small end of the seed was cut and thecotyledons pressed out of the seed coat. Cotyledons were transferred toplates containing SB 199 medium (25-30 cotyledons per plate) for 2weeks, then transferred to SB 1 for 2-4 weeks. Plates were wrapped withfiber tape. After this time, secondary embryos were cut and placed intoSB196 liquid media for 7 days.

Preparation of DNA for Bombardment:

A 50 μL aliquot of sterile distilled water containing 1 mg of goldparticles was added to 5 μL of a 1 μg/μL DNA solution, 50 μL 2.5M CaCl₂and 20 μL of 0.1 M spermidine. The mixture was pulsed 5 times on level 4of a vortex shaker and spun for 5 sec in a bench microfuge. After a washwith 150 μL of 100% ethanol, the pellet was suspended by sonication in85 μL of 100% ethanol. Five μL of DNA suspension was dispensed to eachflying disk of the Biolistic PDS 1000/HE instrument disk. Each 5 μLaliquot contained approximately 0.058 mg gold particles per bombardment(i.e., per disk).

Tissue Preparation and Bombardment with DNA:

Approximately 100-150 mg of 7 day old embryonic suspension cultures wereplaced in an empty, sterile 60×15 mm petri dish and the dish was placedinside of an empty 150×25 mm Petri dish. Tissue was bombarded 1 shot perplate with membrane rupture pressure set at 650 PSI and the chamber wasevacuated to a vacuum of 27-28 inches of mercury. Tissue was placedapproximately 2.5 inches from the retaining/stopping screen.

Selection of Transformed Embryos:

Transformed embryos were selected using hygromycin as the selectablemarker. Specifically, following bombardment, the tissue was placed intofresh SB196 media and cultured as described above. Six to eight dayspost-bombardment, the SB 196 is exchanged with fresh SB 196 containing30 mg/L hygromycin. The selection media was refreshed weekly. Four tosix weeks post-selection, green, transformed tissue was observed growingfrom untransformed, necrotic embryogenic clusters. Isolated, greentissue was removed and inoculated into multi-well plates to generatenew, clonally propagated, transformed embryogenic suspension cultures.

Embryo Maturation:

Transformed embryogenic clusters were cultured for one-three weeks at26° C. in SB 196 under cool white fluorescent (Phillips cool whiteEconowatt F40/CW/RS/EW) and Agro (Phillips P40 Agro) bulbs (40 watt) ona 16:8 hrphotoperiod with light intensity of 90-120 μE/m²s. After thistime embryo clusters were removed to a solid agar media, SB166, for 1week, and then subcultured to medium SB103 for 3 weeks. Alternatively,embryo clusters were removed to SB228 (SHaM) liquid media, 35 mL in 250mL Erlenmeyer flask, for 2-3 weeks. Tissue cultured in SB228 wasmaintained on a rotary shaker, 130 rpm, 26° C. with cool whitefluorescent lights on 16:8 h day/night photoperiod at light intensity of60-85 μE/m2/s. During this period, individual embryos were removed fromthe clusters and screened for alterations in their fatty acidcompositions.

Media Recipes SB 196—FN Lite Liquid Proliferation Medium (Per Liter)

MS FeEDTA - 100x Stock 1 10 mL MS Sulfate - 100x Stock 2 10 mL FN LiteHalides - 100x Stock 3 10 mL FN Lite P, B, Mo - 100x Stock 4 10 mL B5vitamins (1 mL/L) 1.0 mL 2,4-D (10 mg/L final concentration) 1.0 mL KNO₃2.83 gm (NH₄)₂SO₄ 0.463 gm Asparagine 1.0 gm Sucrose (1%) 10 gm pH 5.8

FN Lite Stock Solutions

Stock Number 1000 mL 500 mL 1 MS Fe EDTA 100x Stock Na₂ EDTA* 3.724 g1.862 g FeSO₄—7H₂O 2.784 g 1.392 g 2 MS Sulfate 100x stock MgSO₄—7H₂O37.0 g 18.5 g MnSO₄—H₂O 1.69 g 0.845 g ZnSO₄—7H₂O 0.86 g 0.43 gCuSO₄—5H₂O 0.0025 g 0.00125 g 3 FN Lite Halides 100x Stock CaCl₂—2H₂O30.0 g 15.0 g KI 0.083 g 0.0715 g CoCl₂—6H₂O 0.0025 g 0.00125 g 4 FNLite P, B, Mo 100x Stock KH₂PO₄ 18.5 g 9.25 g H₃BO₃ 0.62 g 0.31 gNa₂MoO₄—2H₂O 0.025 g 0.0125 g

SB1 Solid Medium (Per Liter)

1 package MS salts (Gibco/BRL—Cat. No. 11117-066)

1 mL B5 vitamins 1000× stock

31.5 g Glucose

2 mL 2,4-D (20 mg/L final concentration)

pH 5.7

8 g TC agar

SB 199 Solid Medium (Per Liter)

1 package MS salts (Gibco/BRL—Cat. No. 11117-066)

1 mL B5 vitamins 1000× stock

30 g Sucrose

4 ml 2,4-D (40 mg/L final concentration)

pH 7.0

2 gm Gelrite

SB 166 Solid Medium (Per Liter)

1 package MS salts (Gibco/BRL—Cat. No. 11117-066)

1 mL B5 vitamins 1000× stock

60 g maltose

750 mg MgCl₂ hexahydrate

5 g Activated charcoal

pH 5.7

2 g Gelrite

SB 103 Solid Medium (Per Liter)

1 package MS salts (Gibcol BRL—Cat. No. 11117-066)

1 mL B5 vitamins 1000× stock

60 g maltose

750 mg MgCl2 hexahydrate

pH 5.7

2 g Gelrite

SB 71-4 Solid Medium (Per Liter)

1 bottle Gamborg's B5 salts w/ sucrose (Gibco/BRL—Cat. No. 21153-036)

pH 5.7

5 g TC agar

2,4-D Stock

Obtain premade from Phytotech Cat. No. D 295—concentration 1 mg/mL

B5 Vitamins Stock (Per 100 mL)

Store aliquots at −20° C.

10 g Myo-inositol

100 mg Nicotinic acid

100 mg Pyridoxine HCl

1 g Thiamine

If the solution does not dissolve quickly enough, apply a low level ofheat via the hot stir plate.

SB 228—Soybean Histodifferentiation & Maturation (SHaM) (Per Liter)

DDI H2O 600 ml FN-Lite Macro Salts for SHaM 10X 100 ml MS Micro Salts1000x 1 ml MS FeEDTA 100x 10 ml CaCl 100x 6.82 ml B5 Vitamins 1000x 1 mlL-Methionine 0.149 g Sucrose 30 g Sorbitol 30 g Adjust volume to 900 mLpH 5.8 Autoclave Add to colled media (≦30 C.): *Glutamine (Final conc.30 mM) 4% 100 mL *Note: Final volume will be 101 mL after glutamineaddition.Because glutamine degrades relatively rapidly, it may be preferable toadd immediately prior to using media. Expiration 2 weeks after glutamineis added; base media can be kept longer w/o glutamine.

FN-Lite Macro for SHAM 10×—Stock #1 (Per Liter)

(NH₄)2SO₄ (Ammonium Sulfate) 4.63 g KNO₃ (Potassium Nitrate) 28.3 gMgSO₄*7H₂0 (Magnesium Sulfate Heptahydrate) 3.7 g KH₂PO₄ (PotassiumPhosphate, Monobasic) 1.85 g Bring to volume Autoclave

MS Micro 1000×—Stock #2 (Per 1 Liter)

H₃BO₃ (Boric Acid) 6.2 g MnSO₄*H₂O (Manganese Sulfate Monohydrate) 16.9g ZnSO4*7H20 (Zinc Sulfate Heptahydrate) 8.6 g Na₂MoO₄*2H20 (SodiumMolybdate Dihydrate) 0.25 g CuSO₄*5H₂0 (Copper Sulfate Pentahydrate)0.025 g CoCl₂*6H₂0 (Cobalt Chloride Hexahydrate) 0.025 g KI (PotassiumIodide) 0.8300 g Bring to volume Autoclave

FeEDTA 100×—Stock #3 (Per Liter)

Na₂EDTA* (Sodium EDTA) 3.73 g FeSO₄*7H₂0 (Iron Sulfate Heptahydrate)2.78 g Bring to Volume *EDTA must be completely dissolved before addingiron.Solution is photosensitive. Bottle(s) should be wrapped in foil to omitlight. Autoclave

Ca 100×—Stock #4 (Per Liter)

CaCl₂*2H₂0 (Calcium Chloride Dihydrate) 44 g Bring to Volume Autoclave

B5 Vitamin 1000×—Stock #5 (Per Liter)

Thiamine*HCl 10 g Nicotinic Acid 1 g Pyridoxine*HCl 1 g Myo-Inositol 100g Bring to Volume Store frozen

4% Glutamine—Stock #6 (Per Liter)

DDI water heated to 30° C. 900 ml L-Glutamine 40 g Gradually add whilestirring and applying low heat. Do not exceed 35° C. Bring to VolumeFilter Sterilize Store frozen * * Note: Warm thawed stock in 31° C. bathto fully dissolve crystals.

Oil Analysis:

Somatic embryos were harvested after two weeks of culture in the liquidmaturation medium SB228 (SHaM) liquid media. Approximately 60 and 30events were created in transformations with KS352 and KS394,respectively. All embryos generated for a given event were harvested inbulk and processed as follows. Embryos were frozen on dry ice or byincubation in a −80° C. freezer for two h followed by lyophilization for48 h.

Dried embryos were ground to a fine powder using a genogrinder vial(½″×2″ polycarbonate) and a steel ball (SPEX Centriprep (Metuchen, N.J.,U.S.A.). Grinding time was 30 sec at 1450 oscillations per min. Forevery event, triplicates of approximately 10 mg of tissue were weighedinto Eppendorf tubes, The tissue was extracted using 200 μL heptane atroom temperature under continuous shaking for 2 h. Heptane extracts werecleared by centrifugation and 25 μL of extract was derivatized to fattyacid methyl esters as follows. One mL of a 25% sodium methoxide stocksolution was added to 24 mL of HPLC grade methanol. Sodium methoxide wasstored under an inert gas.

Five μL of a 17:0 TAG (Nu-Chek Prep, Elysian, Minn., USA) stock solution(10 mg/mL) was combined with 25 μl of heptane tissue extract in a glassculture tube, and 500 μL of 1% sodium methoxide was added. Samples werederivatized in a water bath at 50° C. for 15 min. Samples were allowedto cool to RT and 1 mL of 1M NaCl was added followed by brief mixing.FAMEs were extracted into 1 mL of hepatene and 4 μL sample werequantitated by GC analysis.

Results of the oil analysis are presented in Tables 4 and 5. The meanfatty acid methyl ester content and oleic acid content of the embryostransformed with control vector KS 352 were 4.5% and 17.2%,respectively. The corresponding values for embryos transformed with theCA-DGAT1 vector KS 394 were 7.8% and 25.5%, respectively. These resultsdemonstrate that expression of the CA-DGAT1 cDNA of hazelnut increasesoil content and oleic acid content of soybean somatic embryos.

TABLE 4 Esterified Fatty Acid and Oleic Acid Content of Soybean SomaticEmbryos Generated with the No DGAT Control Construct KS 352 KS 352 oleicacid Event FAME (% total # (% DCW) FAME) 22 6.2 18.9 16 5.7 15.8 35 5.619.5 48 5.6 18.5 14 5.5 17.3 43 5.5 18.7 42 5.4 19.3 33 5.3 17.2 68 5.318.6 3 5.2 18.5 4 5.2 18.9 11 5.2 19.1 41 5.2 16.9 51 5.2 18.2 7 5.117.2 10 5.1 19.9 21 5.1 18.2 27 5.1 18.3 1 5 17.6 46 5 18.4 59 5 17.5 665 19.3 5 4.9 15.1 15 4.9 15.7 29 4.9 16.5 2 4.8 17.6 9 4.8 17.4 30 4.817 34 4.8 16.9 19 4.7 14.8 47 4.7 17.4 67 4.7 22.9 13 4.6 17.2 28 4.615.9 39 4.6 18.6 44 4.6 17.1 65 4.6 19.4 6 4.5 13.9 24 4.5 16.1 31 4.515.9 20 4.4 17.1 37 4.4 17.3 69 4.4 19.2 50 4.3 17.2 54 4.3 19.5 55 4.316.1 64 4.3 18.7 32 4.1 14.4 61 4.1 16.8 23 4 16.1 26 4 13.6 49 4 16.518 3.9 16.4 8 3.8 15.5 53 3.8 20.2 63 3.8 17.2 52 3.7 17.3 17 3.6 14.336 3.6 15.7 60 3.4 16.6 12 3.3 15.4 45 3.3 15.2 62 3.3 18.8 40 3.2 13.325 3 12.3 38 3 16.2 57 2.5 18 56 2.3 18.2 58 2.2 16.5 Mean 4.5 17.2

TABLE 5 Esterified Fatty Acid and Oleic Acid Content of Soybean SomaticEmbryos Generated with the CA-DGAT1 Construct KS 394 KS 394 oleic acidEvent FAME (% total # (% DCW) FAME) 2203.2.07 11.9 28.1 2203.1.02 10.329.2 2203.1.03 10.1 26.1 2203.2.17 9.1 28.2 2203.1.01 9.2 26.7 2203.2.138.8 29.0 2203.2.08 8.1 28.6 2203.2.09 8.3 27.5 2203.1.05 8.2 23.12203.2.01 9.1 20.4 2203.2.14 7.4 24.7 2203.2.12 8.0 26.4 2203.2.04 7.928.9 2203.2.02 8.3 29.3 2203.2.15 7.7 20.9 2203.4.03 7.4 24.3 2203.1.047.6 23.6 2203.1.06 7.8 25.3 2203.2.05 8.4 30.0 2203.2.03 8.0 27.92203.2.06 7.7 24.2 2203.2.16 7.5 26.3 2203.2.10 7.3 24.4 2203.4.07 6.926.4 2203.4.01 6.8 25.7 2203.1.07 6.6 25.2 2203.1.08 7.0 24.1 2203.2.116.6 23.2 2203.4.06 6.0 26.7 2203.4.05 6.2 18.9 2203.4.10 5.0 23.02203.5.01 5.1 20.9 Mean 7.8 25.5

Example 4 Creation and Identification of Novel DGAT Genes that Give HighOil Content in Yeast

Libraries of modified DGAT polynucleotides were generated usingrecursive sequence recombination methods (Stemmer, Proc. Natl. Acad.Sci. USA 91: 10747-10751; Ness et. al. Nature Biotechnology 20:1251-1255). These libraries incorporated diversity from related enzymesand also incorporated random changes. The starting polynucleotidesequence in which the diversity was incorporated was CA-DGAT1* (SEQ IDNo:23), which encodes the identical amino acid sequence as CA-DGAT1, andhas a nearly identical nucleotide sequence as CA-DGAT 1 except thatinternal BamH I and EcoR I restriction sites were removed to facilitatecloning. The CA-DGAT1* and novel DGAT genes were cloned into the uniqueBamH I and EcoR I restriction sites of the yeast expression vectorpSZ378 described in Example 1. The CA-DGAT1* gene in the yeastexpression vector is presented as SEQ ID NO: 24. Plasmid PHP35885 (SEQID NO: 151, FIG. 2) is a representative example of a novel DGAT gene,CA-DGAT1-C11, cloned into the yeast expression vector. The libraries ofnovel DGAT genes were amplified in E. coli and then transformed into theSaccharomyces cerevisiae DGAT/PDAT double null strain described inExample 2 using the transformation method of Giest and Schiestl (NatureProtocols 2:38-41) except that the heat shock was done at 37° C. ratherthan 42° C. High oil strains were identified by staining with thefluorescent stain Nile Red (Greenspan et al., J Cell Biol 100: 965-973).A wide variety of Nile Red staining conditions may be used successfully.For example, we usually stained for 5 min a 200 μl volume of a 1:10dilution of a 2 day yeast culture grown in SC minus uracil media. Theyeast was stained with 5 μl of a 0.02 mg/ml Nile Red stock dissolved in95% ethanol. We then read fluorescence intensity using a 489 nmexcitation wavelength and 581 nm emission wavelength. Fluorescenceintensity was divided by absorbance at 600 nm to correct for differencesin cell density. Examples of other conditions used successfully are 3day cultures rather than 2 day, a 1:20 dilution of yeast rather than1:10, staining for 10 min rather than 5 min, having the Nile Red stockdissolved in acetone or 25% DMSO rather than ethanol, and numerousdifferent excitation and emission wavelengths.

Six libraries (libraries A through F) were initially generated andscreened. A second round library J was then generated that combineddiversity from some of the most promising novel DGATs obtained duringthe initial screening. The second round library was also amplified in E.coli, transformed into the Saccharomyces cerevisiae double null strain,and screened by Nile Red as described for the first round libraries.Additional rounds of library generation may include information obtainedduring the first and second rounds of screening as well as furtherdiversity from related enzymes. The additional libraries may be made bycontinuing with the engineered CA-DGAT1* as backbone, or alternativelythe diversity may be generated in a type I DGAT gene from maize,soybean, or another source.

Oil content (total fatty acid methyl esters as a percent of dry weight)and fatty acid composition were determined by quantitative gaschromatography for yeast strains with high Nile Red staining.Approximately 5-15 mg of yeast powder from 2 day cultures were weighedinto the bottom of a 13×100 mm glass culture tube with screw cap andTeflon seal. 5 μL of a stock solution of 17:0 TAG (10 mg/mL in toluene)was added followed by addition of 500 μL 5% sulfuric acid in methanol(anhydrous). Samples were incubated at 95° C. for 1.5 h. Subsequently,tubes were allowed to cool to room temperature after which 1 ml of 1 Msodium chloride was added followed by mixing. One ml of heptane wasadded, contents were mixed and samples were spun briefly to mediatephase separation. Approximately 500 μl of the organic phase wastransferred to a GC vial. Fatty acid methyl esters were analyzed by gaschromatography. Four μl of heptane extract were analyzed on aHewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fusedsilica capillary column (Supelco Inc., Catalog No. 24152). The oventemperature was programmed to hold at 220° C. for 2.7 min, increase to240° C. at 20 C/min and then hold for an additional 2.3 min. Carrier gaswas supplied by a Whatman hydrogen generator. Retention times werecompared to those for methyl esters of standards commercially available(Nu-Chek Prep, Inc. catalog #U-99-A).

Results of the oil analysis are presented in Tables 6, 7 and 8.Palmitic, palmitoleic, stearic, and oleic acid are abbreviated as 16:0,16:1, 18:0, and 18:1, respectively. Higher oil content (total fatty acidmethyl esters as a percent of dry weight) was present in many strainsexpressing novel DGATs in comparison with strains expressing eitherCA-DGAT1*, or a vector control. For unknown reasons, the entire data setof library J, including CA-DGAT1* controls, was lower than observed forthe other libraries, but the novel DGATs from Library J still gavehigher oil than did the CA-DGAT1* controls of this data set. The data ofTables 6, 7, and 8 confirmed that Nile Red staining is indeed effectivein identifying high oil yeast strains, and that novel DGAT genes aremore effective than CA-DGAT1* in increasing oil content in yeast.

TABLE 6 Oil Content and Fatty Acid Composition of Yeast Expressing NovelDGAT Genes from Libraries A, B, and C. DGAT expressed FAME (% dry wt)%16:0 %16:1 %18:0 %18:1 CA-DGAT1-A2 24.2 22.5 41.8 6.6 29.1 CA-DGAT1-A320.8 23.0 41.8 6.9 28.4 CA-DGAT1-C10 20.5 32.9 30.4 12.2 24.6CA-DGAT1-C8 20.5 31.5 31.0 12.2 25.3 CA-DGAT1-C12 19.8 31.6 31.1 12.225.1 CA-DGAT1-C9 19.5 31.8 31.1 12.3 24.8 CA-DGAT1-C11 19.0 31.9 31.012.5 24.7 CA-DGAT1-C13 18.5 31.8 31.0 12.6 24.6 CA-DGAT1-C3 17.9 31.832.1 10.7 25.5 CA-DGAT1-C15 17.6 33.0 30.9 12.2 23.9 CA-DGAT1-A1 17.632.7 31.4 11.6 24.3 CA-DGAT1-C7 17.5 31.8 30.8 12.6 24.8 CA-DGAT1-C1717.3 30.9 31.0 11.8 26.3 CA-DGAT1-C1 16.5 33.5 30.3 12.9 23.2CA-DGAT1-A14 16.4 30.2 33.4 11.8 24.7 CA-DGAT1-C18 16.3 30.7 30.9 11.426.9 CA-DGAT1-C16 16.1 31.1 32.0 10.4 26.6 CA-DGAT1-A9 15.7 29.5 32.512.3 25.7 CA-DGAT1-B6 15.6 31.7 31.6 13.3 23.3 CA-DGAT1-A16 15.3 30.433.0 11.8 24.8 CA-DGAT1-A13 15.2 30.8 32.7 12.6 23.9 CA-DGAT1-A15 15.030.2 34.1 10.0 25.7 CA-DGAT1-A5 14.8 29.7 32.8 12.1 25.5 CA-DGAT1-A714.8 29.1 32.5 12.4 26.0 CA-DGAT1-A17 14.7 29.9 32.6 11.9 25.6CA-DGAT1-C5 14.4 30.6 29.9 12.2 27.3 CA-DGAT1-C14 14.3 30.5 32.1 10.227.1 CA-DGAT1-A10 14.2 30.9 32.4 11.9 24.7 CA-DGAT1-A6 14.2 29.1 32.512.0 26.4 CA-DGAT1-C6 14.1 30.0 26.9 14.4 28.7 CA-DGAT1-A4 14.1 30.932.5 12.6 23.9 CA-DGAT1-A21 13.6 30.5 33.7 10.5 25.3 CA-DGAT1-A23 13.630.1 34.0 11.0 24.9 CA-DGAT1-A22 13.5 29.8 34.1 10.8 25.3 CA-DGAT1-A2013.5 29.7 33.2 11.6 25.5 CA-DGAT1-A24 13.5 29.8 32.8 11.9 25.4CA-DGAT1-A19 13.2 30.6 33.4 11.9 24.1 CA-DGAT1-A12 13.2 29.9 33.0 11.225.9 CA-DGAT1-A8 13.1 28.7 31.9 12.3 27.1 CA-DGAT1-C2 13.0 29.3 28.513.7 28.4 CA-DGAT1-A18 13.0 30.0 32.5 12.1 25.4 CA-DGAT1* 13.0 30.2 32.212.7 24.9 control, rep2 C4 12.9 29.4 28.0 13.2 29.5 B3 12.5 29.5 30.212.9 27.4 A11 11.8 30.5 36.9 9.5 23.1 B8 11.7 30.1 32.1 13.0 24.8 B411.6 27.7 28.9 13.2 30.3 CA-DGAT1* 11.5 29.4 32.5 12.2 26.0 control,rep1 CA-DGAT1-B2 10.6 27.2 29.3 13.1 30.4 CA-DGAT1-B5 10.4 28.5 31.212.6 27.7 CA-DGAT1-B1 10.3 22.3 33.2 12.0 32.5 CA-DGAT1-B9 10.2 26.934.4 10.5 28.2 Vector control 3.1 18.9 39.9 13.8 27.4

TABLE 7 Oil Content and Fatty Acid Composition of Yeast Expressing NovelDGAT Genes from Libraries D, E, and F DGAT expressed FAME (% dry wt)%16:0 %16:1 %18:0 %18:1 CA-DGAT1-D2 19.3 32.6 31.1 12.2 23.4 CA-DGAT1-E418.5 33.1 29.0 13.4 24.5 CA-DGAT1-D16 18.2 32.5 31.4 12.7 23.4CA-DGAT1-E3 18.0 30.2 28.8 13.5 27.4 CA-DGAT1-D19 17.5 32.6 31.7 12.423.2 CA-DGAT1-D15 16.2 32.4 30.7 12.9 24.0 CA-DGAT1-D5 15.8 30.4 27.114.9 27.2 CA-DGAT1-E1 15.3 31.7 35.2 11.8 21.4 CA-DGAT1-F8 15.2 31.832.3 12.5 23.3 CA-DGAT1-E2 15.1 29.6 32.0 11.7 26.6 CA-DGAT1-D14 15.032.2 30.8 12.9 23.9 CA-DGAT1-E5 15.0 31.0 33.3 11.9 23.8 CA-DGAT1-D414.9 29.6 34.5 10.9 24.6 CA-DGAT1-F19 14.8 32.6 32.1 12.5 22.8CA-DGAT1-D20 14.8 31.5 31.1 14.6 22.7 CA-DGAT1-E8 14.8 32.6 30.4 13.323.7 CA-DGAT1-D17 14.5 30.5 33.6 11.3 24.5 CA-DGAT1-E6 14.5 30.9 33.111.6 24.3 CA-DGAT1-D10 14.3 31.3 33.5 12.1 23.0 CA-DGAT1-D9 14.3 30.632.3 11.5 25.5 CA-DGAT1-F5 14.2 32.0 32.8 12.5 22.7 CA-DGAT1-E19 14.231.4 32.7 12.4 23.4 CA-DGAT1-E16 14.0 31.8 32.4 12.7 22.9 CA-DGAT1-E1513.5 32.1 32.5 12.2 23.1 CA-DGAT1-F7 13.4 31.5 32.4 12.2 23.8CA-DGAT1-F18 13.3 32.1 31.6 11.9 24.4 CA-DGAT1-F9 13.3 31.6 33.1 12.422.8 CA-DGAT1-F12 13.3 32.2 32.1 11.7 23.8 CA-DGAT1-E9 13.2 30.8 26.914.8 27.4 CA-DGAT1-D18 13.2 30.1 33.8 11.5 24.5 CA-DGAT1-D12 13.1 30.433.3 11.1 25.1 CA-DGAT1-D7 13.1 30.6 33.9 11.1 24.2 CA-DGAT1-D6 13.129.8 32.8 11.3 25.7 CA-DGAT1-D8 13.1 29.8 34.9 11.3 23.8 CA-DGAT1-E1113.0 29.9 34.2 12.0 23.8 CA-DGAT1-E10 12.8 35.8 26.6 14.5 23.1CA-DGAT1-F20 12.8 31.8 32.2 12.0 24.0 CA-DGAT1-F11 12.8 31.5 33.6 12.122.7 CA-DGAT1-F4 12.7 31.8 32.5 11.4 24.3 CA-DGAT1-E13 12.4 32.4 32.112.0 23.5 CA-DGAT1-F6 12.2 32.2 31.1 12.6 24.1 CA-DGAT1-F17 12.2 32.031.8 12.1 24.2 CA-DGAT1-E18 12.1 31.7 31.9 12.4 24.0 CA-DGAT1-E12 12.029.6 28.3 14.4 27.6 CA-DGAT1-F10 11.7 31.7 32.3 11.7 24.2 CA-DGAT1-F1311.7 31.7 32.4 11.3 24.6 CA-DGAT1-F1 11.4 28.9 35.1 11.2 24.7CA-DGAT1-F16 11.3 31.7 32.0 11.6 24.6 CA-DGAT1-D11 10.7 27.5 33.0 11.727.6 CA-DGAT1-D13 10.6 25.3 33.9 11.7 29.0 CA-DGAT1* 10.6 29.6 34.9 10.624.8 control, rep1 CA-DGAT1-F15 10.5 30.9 32.5 11.2 25.4 CA-DGAT1-F1410.5 31.4 32.1 11.6 24.9 CA-DGAT1-F2 10.4 31.0 32.0 11.8 25.1CA-DGAT1-E14 10.4 28.8 34.3 11.4 25.5 CA-DGAT1-E17 10.4 34.0 30.7 12.822.4 CA-DGAT1-F3 10.2 31.6 31.5 11.8 24.9 CA-DGAT1* 9.7 29.2 35.1 10.725.0 control, rep2 CA-DGAT1-D3 9.4 32.1 29.9 12.0 25.3 CA-DGAT1-D1 7.828.6 22.9 12.0 30.9

TABLE 8 Oil Content and Fatty Acid Composition of Yeast Expressing NovelDGAT Genes from Library J FAME FAME DGAT expressed (% dry wt) (mean ofduplicates) 16:0% 16:1% 18:0% 18:1% CA-DGAT1-J1 11.9 31.9 30.1 13.8 24.1rep1 CA-DGAT1-J1 11.7 11.8 31.8 30.2 13.7 24.3 rep2 CA-DGAT1-J12 14.835.1 31.0 14.4 19.4 rep 1 CA-DGAT1-J12 13.2 14.0 35.1 31.3 14.2 19.4 rep2 CA-DGAT1-J13 13.2 32.1 30.4 14.3 23.1 rep 1 CA-DGAT1-J13 10.6 11.932.5 30.3 14.3 22.9 rep 2 CA-DGAT1-J16 12.8 33.6 29.4 14.4 22.6 rep 1CA-DGAT1-J16 11.5 12.2 33.5 29.4 14.6 22.5 rep 2 CA-DGAT1-J21 14.5 33.330.2 13.7 22.8 rep 1 CA-DGAT1-J21 15.0 14.7 33.6 30.1 13.7 22.6 rep 2CA-DGAT1-J24 10.8 32.1 29.8 14.1 24.0 rep 1 CA-DGAT1-J24 12.4 11.6 32.629.9 13.9 23.6 rep 2 CA-DGAT1-J32 9.0 31.4 30.1 14.4 24.0 rep 1CA-DGAT1-J32 8.1 8.6 31.4 30.4 14.1 24.2 rep 2 CA-DGAT1-J34 12.9 33.429.6 14.4 22.6 rep 1 CA-DGAT1-J34 11.9 12.4 32.6 30.6 13.9 22.9 rep 2CA-DGAT1-J37 10.8 29.3 30.3 13.0 27.4 rep 1 CA-DGAT1-J37 10.0 10.4 29.530.3 12.8 27.4 rep 2 CA-DGAT1-J38 11.6 28.9 33.6 12.4 25.1 rep 1CA-DGAT1-J38 10.3 11.0 28.9 33.2 12.7 25.2 rep 2 CA-DGAT1* 8.3 29.7 33.012.1 25.2 control, rep 1 CA-DGAT1* 7.7 8.0 29.8 32.9 12.1 25.3 control,rep 2

Example 5 Novel DGAT Proteins have Higher Specific Activity thanCA-DGAT1*

Yeast microsomal membrane preparations from some high oil yeast strainsexpressing novel DGAT genes were used for activity assays and westernblots to determine DGAT activity per mg microsomal protein, relativeDGAT protein abundance, and DGAT activity adjusted for DGAT proteinabundance. The activity assays were done as described in Example 2except that 5 μg microsomal protein and 3 μM C14-labeled 18:1-CoA wereused. The endogenous DAG concentration and 3 μM 18:1-CoA appeared to besaturating concentrations for both of these substrates with theCA-DGAT1* enzyme. Western blots were probed with rabbit polyclonalantibodies prepared against the peptide NGNDGGEKIANGEDR (peptide 1, SEQID NO: 176), corresponding to amino acid residues 93 to 107 of theCA-DGAT1 amino acid sequence. This antigenic peptide region wasidentical in all novel DGAT sequences thus far examined (ie. nomutations were present in this region that might affect signal strengthon a western blot). Twenty μl of yeast microsomes were mixed withprotease inhibitors (1 μl Calbiochem Protease Inhibitor Cocktail SetIII, catalog #539134, 1 μl 0.5 M EDTA and 0.5 μl of a 100 mM PMSF stockin isopropanol), and incubated on ice 15 min prior to addition of 2×SDSsample buffer, followed by SDS-PAGE. The Invitrogen I blot dry transfersystem was used to transfer protein to nitrocellulose membrane. Themembrane was rinsed briefly in TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl,0.2% Tween 20), and blocked for 20 min at room temperature in 5% instantnonfat dry milk/TBST with slow rocking. The membrane was then incubated1 h with a 1:2000 dilution of primary antisera in 3% milk/TBST, washed 3times for 5 to 10 min in TBST, and then incubated 1 h with a secondaryantibody consisting of a 1:5000 dilution of goat anti-rabbit IgG-HRPconjugate (Bio-Rad catalog#170-6515) in 3% milk/TBST. Following washingin TBST, the membrane was incubated 5 min with the SuperSignal West DuraECL substrate (Pierce catalog #34075). The blots were imaged using aFujifilm LAS3000 imaging system. Pixel densities for the target bandswere determined with TotalLab's software, Nonlinear TL120 v2006e. Forcontrol samples on each blot, several concentrations of microsomalprotein from yeast expressing CA-DGAT1* were used. The relativeabundance of novel DGAT protein present in microsomes was calculated bycomparing with the CA-DGAT1* control microsome samples.

Results of the activity assays and western blots are reported in TABLE9. As evident in the right column of TABLE 9, 14 of the 19 novel DGATsexamined had greater activity than CA-DGAT1* following adjustment forDGAT protein abundance. Three novel DGATs (CA-DGAT1-C9, CA-DGAT1-C11,and CA-DGAT1-E3) had adjusted activities more than 3-fold greater thanthat of CA-DGAT1*. Such large increases in DGAT specific activity as aresult of mutagenesis have not been reported previously. One novel DGAT(CA-DGAT1-J21) had similar adjusted activity, and only 4 (CA-DGAT1-C10,CA-DGAT1-D2, CA-DGAT1-D16, and CA-DGAT1-J16) had lower adjusted activitythan CA-DGAT1*. In most cases, the engineered DGAT proteins were lessabundant in microsomes than was the CA-DGAT1* protein. Less abundancemay be due to either lower expression level in yeast or due to the factthat proportionately more of the novel DGAT protein was in the fat pad,rather than the microsomal pellet, compared with the CA-DGAT1* protein.Some of the yeast strains expressing novel DGATs had extremely large fatpads evident during microsomal preparation, consistent with the veryhigh oil contents reported in TABLES 6, 7, and 8.

TABLE 9 Activity and Relative Abundance of Novel DGAT from High OilYeast Strains DGAT Activity DGAT Activity Adjusted for DGAT (pmol C14labeled Abundance (pmol oleoyl-CoA incor- DGAT Abundance C14 labeledoleoyl- DGAT Activity porated into TAG in Microsomes CoA incorporatedAdjusted for DGAT per minute per mg relative to into TAG per minute perAbundance DGAT microsomal protein) CA-DGAT1* mg microsomal protein) (%of CA-DGAT1*) CA-DGAT1* 3596 1 3596 100 A2 6184 0.92 6722 187 C9 21690.18 12050 335 C10 2307 0.83 2780 77 C11 2362 0.2 11810 328 C13 18800.29 6483 180 D2 2809 1.25 2247 62 D16 1891 0.95 1991 55 D19 3025 0.65042 140 E3 2285 0.2 11425 318 J1 3128 0.73 4285 119 J12 2738 0.63 4346121 J13 3384 0.64 5288 147 J16 1753 0.55 3187 89 J21 2797 0.76 3680 102J24 2778 0.54 5144 143 J32 1266 0.25 5064 141 J34 2742 0.58 4728 131 J371951 0.32 6097 170 J38 4142 0.79 5243 146

Example 6 Novel DGAT Genes Give Significantly Higher Oil and Oleic AcidContents in Soybean Somatic Embryos than does CA-DGAT1*

Four novel DGAT genes (CA-DGAT1-A2, CA-DGAT1-C9, CA-DGAT1-C10, andCA-DGAT1-C11) that gave high oil when expressed in yeast, plus theCA-DGAT1* gene, were ectopically expressed in soybean somatic embryosunder control of the soybean β-conglycinin α′ subunit promoter using themethods of EXAMPLE 3.

Oil content of soybean somatic embryos was determined by NMR using aMaran Ultra NMR analyzer (Resonance Instruments Ltd, Whitney,Oxfordshire, UK). Samples were placed into pre-weighed 2 mLpolypropylene tubes (Corning Inc, Corning N.Y., USA; Part no. 430917)previously labeled with unique bar code identifiers. Samples were thenplaced into 96 place carriers and processed through the following seriesof steps by an Adept Cobra 600 SCARA robotic system.

-   -   1. pick up tube (the robotic arm was fitted with a vacuum pickup        devise)    -   2. read bar code    -   3. expose tube to antistatic device    -   4. weigh tube (containing the sample), to 0.0001 g precision.    -   5. NMR reading; measured as the intensity of the proton spin        echo 1 msec after a 22.95 MHz signal had been applied to the        sample (data was collected for 32 NMR scans per sample)    -   6. return tube to rack    -   7. repeat process with next tube        Bar codes, tube weights and NMR readings were recorded by a        computer connected to the system. Sample weight was determined        by subtracting the polypropylene tube weight from the weight of        the tube containing the sample.

Oil content was calculated as follows:

${\% \mspace{14mu} {oil}\mspace{11mu} ( {\% \mspace{14mu} {wt}\mspace{14mu} {basis}} )} = \frac{ {( {{NMR}\mspace{14mu} {{signal}/{sample}}\mspace{14mu} {wt}\mspace{11mu} (g)} ) - 70.58} )}{351.45}$

Calibration parameters were determined by precisely weighing samples ofsoy oil (ranging from 0.0050 to 0.0700 g at approximately 0.0050 gintervals; weighed to a precision of 0.0001 g) into Corning tubes (seeabove) and subjecting them to NMR analysis. A calibration curve of oilcontent to NMR value was established.

Results of the oil analysis are presented in TABLE 10 and FIG. 3.Looking at mean values, all 4 novel DGAT genes gave higher oil than didthe CA-DGAT1* gene when expressed in soybean somatic embryos. TheCA-DGAT1-C11, CA-DGAT1-C10, and CA-DGAT1-C9 values of 9.0, 8.6, and 7.6%oil were considerably greater than the CA-DGAT1* value of 5.5% oil.

TABLE 10 Oil Content of Soybean Somatic Embryos Expressing Novel DGATGenes or CA-DGAT1* % Oil CA-DGAT1* CA-DGAT1-A2 CA-DGAT1-C9 CA-DGAT1-C10CA-DGAT1-C11 9.3 7.7 11.5 14.2 13.3 9.3 7.5 10.9 13.5 13.2 8.8 7.4 10.812.6 13.1 8.1 7.4 10.6 11.5 12.7 7.3 7.2 10.6 10.8 12.0 7.1 7.2 10.410.8 11.9 7.0 7.1 9.6 10.7 11.2 6.8 7.1 9.3 10.6 11.0 6.4 7.1 8.9 10.010.8 6.3 7.1 8.5 10.0 10.8 6.3 6.5 8.1 9.8 10.2 5.6 6.4 8.1 9.7 9.9 5.56.3 7.9 9.5 9.3 5.4 6.2 7.7 9.3 9.3 5.4 6.1 7.7 9.3 9.2 5.1 6.1 7.6 9.09.2 4.9 6.1 7.4 9.0 8.7 4.7 6.1 7.2 8.9 8.4 4.6 5.8 7.0 8.7 8.4 4.6 5.86.7 8.2 7.9 4.5 5.7 6.2 8.1 7.9 4.1 5.7 6.1 7.9 7.2 4.1 5.3 5.9 6.8 7.14.1 5.2 5.5 6.3 7.0 4.1 4.9 5.5 5.9 6.9 4.0 4.5 5.3 5.7 6.8 4.0 4.4 5.15.4 6.4 3.9 4.1 5.1 4.6 5.8 3.7 3.5 4.9 4.0 5.5 3.5 3.5 4.8 3.9 4.3 3.43.3 4.5 3.1 4.2 Mean. 5.5 6.0 7.6 8.6 9.0 Mean of 7.6 7.3 10.1 11.5 12.0top 10.

Fatty acid composition of the soy somatic embryos was determined by gaschromatography for the top 4 events of each of the 4 novel DGAT genesand the CA-DGAT1* gene (TABLE11). Expression of each novel DGAT resultedin more 18:1 and 18:0, and less 18:2 and 16:0, than observed withexpression of CA-DGAT1*. Novel DGAT gene expression also resulted inless 18:3 than observed with CA-DGAT1* expression, with the exception ofnovel DGAT A2.

TABLE 11 Fatty acid composition of soybean somatic embryos expressingCA-DGAT1* or novel DGAT genes. DGAT %16:0 %18:0 %18:1 %18:2 %18:3CA-DGAT1* 15.2 4.4 21.0 49.1 10.3 CA-DGAT1* 17.5 4.0 15.5 51.8 11.3CA-DGAT1* 16.9 3.9 17.6 49.9 11.6 CA-DGAT1* 14.2 5.4 23.6 45.0 11.8 Meanof CA- 16.0 4.4 19.4 48.9 11.3 DGAT1* CA-DGAT1-A2 15.3 4.6 22.4 46.511.2 CA-DGAT1-A2 14.4 5.9 25.1 42.0 12.7 CA-DGAT1-A2 14.3 6.2 23.4 43.612.5 CA-DGAT1-A2 15.3 6.0 27.2 40.1 11.4 Mean of CA- 14.8 5.7 24.5 43.011.9 DGAT1-A2 CA-DGAT1-C9 12.9 6.8 32.2 40.0 8.1 CA-DGAT1-C9 14.5 4.620.9 49.9 10.0 CA-DGAT1-C9 14.1 6.0 27.7 43.3 8.9 CA-DGAT1-C9 14.0 6.330.5 40.0 9.1 Mean of CA- 13.9 5.9 27.8 43.3 9.0 DGAT1-C9 CA-DGAT1-C1013.6 5.5 29.6 44.1 7.3 CA-DGAT1-C10 13.6 5.4 28.6 44.9 7.5 CA-DGAT1-C1013.4 5.0 30.0 44.1 7.5 CA-DGAT1-C10 14.3 6.4 25.6 44.9 8.8 Mean of CA-13.7 5.6 28.5 44.5 7.8 DGAT1-C10 CA-DGAT1-C11 14.3 4.3 23.8 48.7 8.8CA-DGAT1-C11 13.5 4.8 29.2 43.4 9.2 CA-DGAT1-C11 13.2 5.2 29.5 43.5 8.7CA-DGAT1-C11 14.2 4.9 26.9 45.6 8.5 Mean of CA- 13.8 4.8 27.4 45.3 8.8DGAT1-C11The data of TABLES 10 and 11 demonstrate that novel DGAT genesidentified by screening for high oil in yeast give higher oil and oleicacid contents when expressed in soybean somatic embryos than does theCA-DGAT1* gene. Additional novel DGAT genes may be tested by ectopicexpression in soybean somatic embryos, and the most promising novel DGATgenes may be expressed in soybean seeds to provide increases in oil andoleic acid content.

Example 7 Sequences of Novel DGAT Genes Giving High Oil Content in Yeast

DNA sequences were determined for 63 of the novel DGAT genes that gavehigher oil content in yeast than that obtained with CA-DGAT1*. Thecorresponding amino acid sequences were deduced from the DNA sequences.The DNA and amino acid sequences of the novel DGATs are presented as SEQID NO: 25 to SEQ ID NO: 150. A partial summary of the amino acidsubstitutions can observed in the alignment of DGAT sequences presentedin FIG. 5. The mean number of substitutions observed in the 63 novelDGATs was 10, and the maximum number was 19 (observed in CA-DGAT1-J34).Only 2 of the 63 novel DGATS had less than 4 amino acid substitutions,with CA-DGAT1-F19 having 1, and CA-DGAT1-F8 having 2. Considering all 63novel DGATs, 115 different substitutions were present at a total of 104different positions (ie. 2 kinds of substitutions were present at 11positions). Of the 115 different substitutions, 53 were observed onlyonce, 62 were observed at least twice, 56 were observed at least 3times, and 50 were observed at least 4 times. The most frequentlyobserved substitution was F514S, which was present in 36 of the 63 novelDGATs.

The 115 different substitutions observed in novel DGAT sequences are:D5G, P7L, G11D, T12A, T18A, H30P, N31A, E34A, T45A, T46A, P48S, P48A,D49S, D49N, G51D, N52D, V54K, V58A, R59T, D66S, S68A, S68P, S73T, S73G,S79N, R80K, E86N, E86D, S87N, N93S, G97D, T109M, A112T, K115Q, Y118L,P123A, A124T, I128V, S135T, F139L, F139S, H143R, L146P, V1551, I162V,K174E, S181A, S181T, R182G, L184S, M191V, P197V, I198V, F199L, F204S,V205M, Q211T, K213R, P218H, L222V, R241K, L247V, T251V, M253V, A256S,C257S, Y266S, T267A, D281E, N288D, N288S, D293E, Y294N, S299T, K301N,M307V, V308L, 1324V, S327G, V329L, V329M, Q332R, V334L, I337V, I338V,I346M, E348K, K356Q, K363N, Y368H, A369V, L385F, C390S, L399V, E409G,K412E, H432Y, M435V, V4361, V436R, G451A, A457S, V460A, 1475M, C486F,V488I, V491L, R502K, S504T, N₅O₈S, F514S, L518V, L531V, N533S, andN533D.

Out of the 115 total substitutions observed, the 62 substitutions thatwere observed at least twice are: T12A, T18A, H30P, N31A, P48S, D49S,G51D, N52D, R59T, D66S, S68A, S73T, S79N, E86N, S87N, Y118L, P123A,1128V, F139L, V1551, S181A, L184S, P197V, I198V, V205M, Q211T, P218H,L222V, R241K, L247V, T251V, A256S, C257S, Y266F, T267A, D281E, N288D,D293E, Y294N, S299T, K301N, V308L, S327G, V329L, V334L, I337V, I338V,K356Q, K363N, C390S, L399V, V4361, G451A, A457S, I475M, C486F, V488I,V491L, R502K, F514S, L518V, and L531V,

Example 8 Transferring Amino Acid Substitutions from Novel Hazelnut DGATinto Soybean DGAT

The novel DGAT genes CA-DGAT1-C9, CA-DGAT1-C10, and CA-DGAT1-C11 werevery effective in increasing oil and oleic content in soybean somaticembryos (TABLES 10 and 11). Therefore, some of the amino acidsubstitutions present in these highly effective novel hazel DGATs weretransferred to soybean DGAT to determine whether these samesubstitutions could increase the effectiveness of soybean DGAT forincreasing oil or oleic content. The substitutions that were transferredare summarized in TABLE 12. The soybean DGAT gene with no substitutionsis named GM-DGAT1, and the corresponding DNA and deduced amino acidsequences are presented as SEQ ID NO: 152 and 153. The four novelsoybean DGAT genes are named GM-DGAT1-C9 (SEQ ID NO: 154 and 155),GM-DGAT1-C10 (SEQ ID NO: 156 and 157), GM-DGAT1-C11 (SEQ ID NO: 158 and159) and GM-DGAT1-C9C10C11 (SEQ ID NO: 160 and 161). These four novelsoybean DGAT proteins contained 5, 5, 11, and 14 amino acidsubstitutions, respectively.

TABLE 12 Amino Acid Substitutions in Novel Hazelnut DGAT and theCorresponding Substitutions Made in Soybean DGAT Some substitutionsobserved in novel hazelnut DGAT genes Substitutions SubstitutionsSubstitutions Substitutions CA-DGAT1-C9, made in novel made in novelmade in novel made in novel CA-DGAT1-C10, soy DGAT soy DGAT soy DGAT soyDGAT or CA-DGAT1-C11 GM-DGAT1-C9 GM-DGAT1-C10 GM-DGAT1-C11GM-DGAT1-C9C10C11 S79N S58N S58N S181A S146A S146A S146A S146A V205MI170M I170M R241K R206K R206K R206K Y266F Y231F Y231F D293E D258E D258ES299T S264T S264T V308L V273L V273L V273L I338V I303V I303V L399V L364VL364V L364V A457S A422S A422S I475M I440M I440M R502K R467K R467K L518VL483V L483V L483V L483V

GM-DGAT1-C9 (SEQ ID NO: 154 and 155), GM-DGAT1-C10 (SEQ ID NO: 156 and157), GM-DGAT1-C11 (SEQ ID NO: 158 and 159) and GM-DGAT1-C9C10C11 (SEQID NO: 160 and 161) were synthesized by GENEART AG (Regensburg, Germany)with NotI restriction enzyme sites flanking the codon-optimized genesequence (before start codon and after stop codon). In addition, threenucleotides (ACC) were added between the NotI at the 5′ end ofcodon-optimized gene and the ATG start codon in all cases.

The NotI fragments containing each synthesized DGAT gene sequence werecloned into the NotI site of soybean expression vector pKR72 (SEQ IDNO:178; described in PCT Publication WO/04071467, published on Aug. 26,2004) which contains a NotI restriction site, flanked by the promoterfor the α′ subunit of β-conglycinin (Beachy et al., EMBO J. 4:3047 3053(1985)) and the 3′ transcription termination region of the phaseolingene (Doyle et al., J. Biol. Chem. 261:9228-9238 (1986)), thus allowingfor strong tissue-specific expression in the seeds of soybean of genescloned into the NotI site. The vector sequences containing wild-type andmutated sequences are summarized in Table 13.

Soybean embryogenic suspension cultures (cv. Jack) were transformed withthe vectors described in Table 13, events were selected and somaticembryos were matured as described in Example 3. Experiment numbers foreach experiment are also summarized in Table 13.

TABLE 13 Summary of wild-type and mutant GmDGAT1s and respective soybeanexpression vectors and corresponding experiment names nt aa ExpressionVector Experiment Gene SEQ ID NO: SEQ ID NO: Vector SEQ ID NO: NumberGM-DGAT1 152 153 pKR1466 179 MSE2515 GM-DGAT1-C9 154 155 pKR1515 180MSE2516 GM-DGAT1-C10 156 157 pKR1516 181 MSE2517 GM-DGAT1-C11 158 159pKR1517 182 MSE2518 GM-DGAT1-C9C10C11 160 161 pKR1520 183 MSE2519

Approximately 30 events for each experiment were created intransformations with the vectors described in Table 13. All embryosgenerated for a given event were harvested in bulk and processed asfollows. Embryos were frozen on dry ice or by incubation in a −80° C.freezer for two h followed by lyophilization for 48 h. Dried embryoswere ground to a fine powder using a genogrinder vial (½″×2″polycarbonate) and a steel ball (SPEX Centriprep (Metuchen, N.J.,U.S.A.). Grinding time was 30 sec at 1450 oscillations per min.

For analysis of fatty acids, a small scoop (˜5 mg) of pulverized powderfor each event was transferred to a glass GC vial. Fortransesterification, 50 μL of trimethylsulfonium hydroxide (TMSH) and0.5 mL of hexane were added to each vial and incubated for 30 min atroom temperature while shaking. Fatty acid methyl esters (1 μL injectedfrom hexane layer) were separated and quantified using a Hewlett-Packard6890 Gas Chromatograph fitted with an Omegawax 320 fused silicacapillary column (Catalog #24152, Supelco Inc.). The oven temperaturewas programmed to hold at 220° C. for 2.6 min, increase to 240° C. at20° C./min and then hold for an additional 2.4 min. Carrier gas wassupplied by a Whatman hydrogen generator. Retention times were comparedto those for methyl esters of standards commercially available (Nu-ChekPrep, Inc.).

Oil concentration measurements for each event from each experiment weredetermined on the remaining dried embryo powders (−20-200 mg each) usingNMR as described in Example 6. Oil concentration and fatty acid profile(fatty acid concentration expressed on a wt. % of total fatty acids)results for MSE2515, MSE 2516, MSE2517, MSE2518 and MSE2519 are shown inTables 14, 15, 16, 17 and 18, respectively. Also shown are the mean oilconcentrations (avg.) and mean fatty acid concentrations (expressed as awt. % of total fatty acids) for all events as well as the meanconcentrations for the top five events having highest oil concentrations(Top5 avg.).

TABLE 14 Oil concentrations and fatty acid profiles for events fromMSE2515 MSE2515 (GmDGAT1) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2515-1416.7 5.6 23.8 38.4 15.3 6.4 2515-13 17.0 5.3 21.7 39.5 16.5 5.9 2515-616.8 5.5 23.2 37.7 16.9 5.1 2515-11 17.5 5.4 23.0 37.8 16.2 5.0 2515-1917.9 4.9 18.1 40.9 18.2 4.8 2515-28 17.3 5.1 16.0 42.5 19.1 4.7 2515-216.9 5.2 21.3 39.5 17.2 4.6 2515-10 17.6 5.0 16.6 42.2 18.6 4.6 2515-2415.9 5.0 18.6 39.9 20.6 4.4 2515-20 17.9 4.8 17.7 39.2 20.4 4.3 2515-1717.0 6.3 20.5 36.9 19.2 4.3 2515-12 17.4 5.1 17.2 38.7 21.7 4.2 2515-718.7 4.4 14.1 42.3 20.5 4.2 2515-29 16.5 4.6 15.9 38.6 24.4 4.1 2515-1518.0 5.1 16.8 42.5 17.7 4.1 2515-9 17.8 4.3 14.3 42.5 21.2 4.0 2515-2217.2 5.5 18.9 39.9 18.5 4.0 2515-5 17.6 4.6 16.6 39.5 21.7 4.0 2515-419.2 5.5 14.8 36.9 23.6 3.9 2515-18 18.0 6.1 20.9 38.2 16.9 3.9 2515-1616.5 4.8 19.1 38.2 21.4 3.9 2515-8 17.4 5.0 17.2 40.4 20.0 3.8 2515-2117.1 4.2 14.0 39.6 25.1 3.8 2515-27 16.0 4.4 17.5 40.3 21.8 3.6 2515-3117.6 4.5 13.0 40.9 24.0 3.6 2515-25 17.0 6.3 19.7 37.1 20.0 3.6 2515-2618.0 4.5 15.2 39.4 22.9 3.5 2515-23 17.4 4.7 14.8 40.9 22.2 3.2 2515-118.4 4.2 13.5 41.7 22.2 3.2 2515-30 16.3 4.0 15.1 40.7 24.0 2.9 2515-320.1 4.5 10.7 41.1 23.5 2.5 Avg. 17.4 5.0 17.4 39.8 20.4 4.1 Top5 Avg.17.2 5.3 22.0 38.9 16.6 5.4

TABLE 15 Oil concentrations and fatty acid profiles for events fromMSE2516 MSE2516 (GmDGAT1-C9) Event 16:0 18:0 18:1 18:2 18:3 % Oil 2516-816.3 7.1 26.4 36.5 13.7 7.1 2516-28 16.9 6.9 25.2 36.8 14.3 6.7 2516-416.0 7.0 24.5 37.1 15.3 6.6 2516-16 16.0 5.4 17.8 45.9 14.8 6.2 2516-1916.4 6.0 24.8 37.6 15.1 6.1 2516-25 17.2 5.4 19.3 39.4 18.7 5.7 2516-2716.1 6.5 22.3 38.6 16.4 5.7 2516-6 17.6 5.3 17.2 38.7 21.1 4.8 2516-117.2 6.3 20.7 37.9 17.9 4.8 2516-30 16.3 6.2 21.0 38.9 17.7 4.7 2516-1315.6 5.6 19.5 39.4 19.9 4.5 2516-22 17.1 5.2 17.8 39.9 19.9 4.3 2516-2617.1 6.0 20.2 38.1 18.6 4.3 2516-12 17.7 5.7 18.8 39.1 18.8 4.3 2516-516.9 5.3 16.2 41.7 20.0 4.2 2516-15 17.6 3.8 15.2 42.1 21.3 4.1 2516-2916.2 5.1 18.8 38.6 21.2 3.9 2516-9 17.3 5.8 19.9 39.0 18.1 3.8 2516-2316.8 5.6 18.9 37.3 21.3 3.8 2516-3 16.6 5.3 17.4 39.8 21.0 3.7 2516-2116.6 6.0 19.5 38.0 19.9 3.6 2516-10 16.3 4.5 15.5 39.9 23.8 3.6 2516-216.5 5.4 17.3 38.0 22.9 3.5 2516-11 17.7 4.9 14.2 41.6 21.6 3.5 2516-1816.5 5.5 18.6 38.9 20.6 3.5 2516-7 19.0 5.0 14.7 38.8 22.5 3.4 2516-2417.9 6.2 20.7 36.6 18.6 3.2 2516-17 17.0 4.2 13.4 41.2 24.2 2.9 Avg.16.9 5.6 19.1 39.1 19.3 4.5 Top5 Avg. 16.3 6.5 23.8 38.8 14.6 6.5

TABLE 16 Oil concentrations and fatty acid profiles for events fromMSE2517 MSE2517 (GmDGAT1-C10) Event 16:0 18:0 18:1 18:2 18:3 % Oil2517-4 15.1 5.8 28.1 40.6 10.4 10.5 2517-15 15.9 5.8 26.3 39.4 12.6 9.12517-7 16.1 5.5 25.4 40.9 12.0 9.0 2517-9 18.0 5.7 23.5 41.0 11.8 8.22517-17 15.9 7.2 30.7 34.8 11.4 7.8 2517-20 17.2 5.9 22.4 40.1 14.5 7.22517-27 18.2 4.3 17.0 43.7 16.7 6.3 2517-8 16.6 6.3 25.4 37.5 14.1 6.02517-19 16.5 4.4 17.0 44.0 18.3 5.7 2517-1 16.4 5.3 17.4 42.4 18.5 5.52517-12 17.0 5.1 18.4 42.6 17.0 5.2 2517-23 16.6 5.6 23.1 38.8 15.9 4.92517-28 17.2 5.2 23.5 36.4 17.6 4.9 2517-5 16.2 5.1 18.7 40.8 19.1 4.92517-14 18.6 6.3 22.2 36.9 15.9 4.8 2517-18 17.4 6.0 20.8 39.1 16.8 4.22517-2 18.6 4.7 13.9 42.6 20.3 4.1 2517-21 16.3 4.6 20.1 40.4 18.6 4.02517-10 16.6 6.6 26.5 33.8 16.4 3.9 2517-24 18.8 5.7 19.7 38.1 17.7 3.82517-31 16.5 4.2 19.1 39.5 20.8 3.7 2517-26 18.7 4.2 12.7 40.1 24.3 3.62517-25 18.1 5.2 17.5 40.5 18.6 3.6 2517-6 17.0 4.3 16.0 40.8 21.9 3.32517-11 17.7 4.6 15.1 40.5 22.1 3.3 2517-29 17.3 4.5 15.9 40.1 22.1 3.12517-16 17.5 3.9 11.3 42.4 24.9 3.0 2517-13 17.6 5.0 18.8 37.4 21.1 2.62517-30 18.0 4.6 15.5 39.5 22.4 2.5 2517-22 17.1 4.4 14.9 39.8 23.7 2.4Avg. 17.2 5.2 19.9 39.8 17.9 5.0 Top5 Avg. 16.2 6.0 26.8 39.3 11.7 8.9

TABLE 17 Oil concentrations and fatty acid profiles for events fromMSE2518 MSE2518 (GmDGAT1-C11) Event 16:0 18:0 18:1 18:2 18:3 % Oil2518-30 15.7 7.6 29.0 35.1 12.6 7.6 2518-18 15.9 8.6 31.4 33.6 10.4 7.52518-3 15.9 6.3 25.1 38.4 14.3 6.6 2518-4 16.7 7.0 24.1 36.5 15.7 6.52518-12 16.2 6.2 24.0 38.0 15.6 6.4 2518-17 15.5 5.6 24.0 38.5 16.3 6.22518-11 16.5 5.5 19.2 40.8 17.9 5.9 2518-23 16.2 6.7 24.1 36.6 16.4 5.92518-19 15.6 5.7 19.5 41.1 18.0 5.6 2518-21 16.6 6.4 21.8 37.6 17.7 5.42518-9 16.8 6.5 23.1 37.0 16.6 5.2 2518-27 15.3 5.9 21.1 39.6 18.2 5.22518-14 16.9 5.6 19.9 39.5 18.1 5.1 2518-2 16.9 5.1 19.2 40.0 18.7 5.02518-26 17.0 5.0 15.4 41.4 21.2 4.5 2518-16 17.8 4.0 12.5 39.2 26.5 4.32518-24 17.1 5.7 18.2 38.3 20.7 4.0 2518-13 16.4 5.7 18.5 39.0 20.4 4.02518-22 17.3 4.9 17.6 38.9 21.4 3.9 2518-8 16.9 6.6 23.0 36.4 17.1 3.92518-5 15.9 5.2 17.0 40.5 21.4 3.7 2518-6 17.0 4.6 13.9 40.6 23.9 3.72518-15 16.5 4.8 16.6 39.2 23.0 3.7 2518-25 16.8 5.2 18.2 39.8 19.9 3.52518-10 17.1 6.5 22.3 35.9 18.2 3.5 2518-29 16.9 5.6 17.9 38.7 21.0 3.42518-20 18.1 5.2 16.6 37.5 22.6 3.3 Avg. 16.6 5.8 20.5 38.4 18.7 4.9Top5 Avg. 16.1 7.2 26.7 36.3 13.7 6.9

TABLE 18 Oil concentrations and fatty acid profiles for events fromMSE2519 MSE2519 (GmDGAT1-C9C10C11) Event 16:0 18:0 18:1 18:2 18:3 % Oil2519-17 16.8 8.6 30.4 35.3 8.9 11.3 2519-20 15.2 6.3 28.1 39.6 10.8 11.22519-9 15.5 7.2 29.7 36.9 10.7 10.5 2519-26 15.9 7.0 28.6 37.6 11.0 10.32519-1 15.5 7.3 28.4 37.5 11.3 10.0 2519-5 15.7 7.8 29.1 36.7 10.8 9.82519-23 15.4 6.1 26.6 40.4 11.6 9.5 2519-10 15.5 6.6 27.3 38.4 12.2 9.42519-16 16.5 6.3 25.4 39.4 12.3 9.3 2519-3 14.9 8.2 29.5 36.6 10.8 8.82519-11 15.8 5.1 21.8 43.9 13.4 8.6 2519-8 14.8 7.6 24.4 41.5 11.8 8.52519-27 15.4 6.5 25.7 39.9 12.4 8.4 2519-19 15.8 7.9 27.5 36.3 12.5 8.12519-18 15.9 7.3 25.5 37.9 13.5 7.1 2519-24 15.5 5.8 20.5 41.0 17.1 6.02519-13 17.2 5.3 19.4 40.4 17.7 5.9 2519-28 16.0 6.2 22.3 38.7 16.7 5.82519-21 15.9 6.1 20.4 39.2 18.4 4.8 2519-30 16.9 6.5 20.7 38.9 17.1 4.72519-29 16.7 5.6 19.9 39.9 17.9 4.6 2519-25 17.0 6.0 19.7 38.8 18.5 4.42519-2 17.2 6.2 24.7 37.3 14.6 4.4 2519-12 16.5 5.3 17.8 39.7 20.7 4.32519-6 16.1 4.4 14.7 41.6 23.3 4.1 2519-4 17.0 5.5 19.0 39.3 19.3 4.02519-22 16.0 4.7 15.9 42.0 21.3 3.7 2519-7 17.0 5.3 16.1 39.3 22.3 3.62519-15 17.0 6.5 20.8 36.8 18.8 3.2 2519-14 18.2 4.4 12.6 40.0 24.9 2.7Avg. 16.2 6.3 23.1 39.0 15.4 6.9 Top5 Avg. 15.8 7.3 29.0 37.4 10.5 10.7

The average oil concentrations for all events for MSE2515, MSE2516,MSE2517, MSE2518 and MSE2519 are 4.1%, 4.5%, 5.0%, 4.9% and 6.9%,respectively. The average oil concentrations for the top five eventshaving highest oil concentrations for MSE2515, MSE2516, MSE2517, MSE2518and MSE2519 are 5.4%, 6.5%, 8.9%, 6.9% and 10.7%, respectively.

Oil concentration plotted versus oleic acid concentration for MSE2515,MSE 2516, MSE2517, MSE2518 and MSE2519 is shown in FIG. 6.

Tables 14-18 and FIG. 6, show that GmDGAT1-C9 and GmDGAT1-C11 expressioncause a small increase in oil and oleic acid concentrations in somaticembryos over that observed for GmDGAT1. GmDGAT1-C10 had a larger effectand GmDGAT1-C9C10C11 had the largest effect on oil and oleic acidconcentrations compared to GmDGAT1 in somatic embryos.

Example 9 Ectopic Expression of Novel DGAT Genes in Soybean Seed

The method for expressing DGAT genes in soybean somatic embryos undercontrol of the soybean β-conglycinin α′ subunit promoter was describedin EXAMPLE 3. The present example describes the method for expressingDGAT genes in soybean seed, rather than somatic embryos.

Transgenic soybean lines are generated by the method of particle gunbombardment (Klein et al., Nature (London) 327:70-73 (1987); U.S. Pat.No. 4,945,050) using a BIORAD Biolistic PDS1000/He instrument andplasmid DNA as described in EXAMPLE 3. The following stock solutions andmedia are used for transformation and regeneration of soybean plants:

Stock Solutions: Sulfate 100× Stock:

37.0 g MgSO₄.7H₂O, 1.69 g MnSO₄.H₂O, 0.86 g ZnSO₄.7H₂O, 0.0025 g

CuSO₄.5H₂O

Halides 100× Stock:

30.0 g CaCl₂.2H₂O, 0.083 g KI, 0.0025 g CoCl₂.6H₂O

P, 13, Mo 100× Stock:

18.5 g KH₂PO₄, 0.62 g H₃BO₃, 0.025 g Na₂MoO₄.2H₂O

Fe EDTA 100× Stock:

3.724 g Na₂ EDTA, 2.784 g FeSO₄.7H₂O

2,4-D Stock:

10 mg/mL Vitamin B5 1000× Stock: 10.0 g myo-inositol, 0.10 g nicotinicacid, 0.10 g

pyridoxine HCl, 1 g thiamine.

Media (Per Liter):

SB196: 10 mL of each of the above stock solutions, 1 mL B5 Vitaminstock, 0.463 g (NH₄)₂ SO₄, 2.83 g KNO₃, 1 mL 2,4-D stock, 1 gasparagine, 10 g Sucrose, pH 5.7

SB103:

1 pk. Murashige & Skoog salts mixture, 1 mL B5 Vitamin stock, 750 mgMgCl₂

hexahydrate, 60 g maltose, 2 g gelrite, pH 5.7.

SB166:

SB103 supplemented with 5 g per liter activated charcoal.

SB71-4:

Gamborg's B5 salts, 1 mL B5 vitamin stock, 30 g sucrose, 5 g TC agar, pH5.7.

To prepare tissue for transformation, soybean embryogenic suspensioncultures are maintained in 35 mL liquid medium (SB196) on a rotaryshaker (150 rpm) at 28° C. with fluorescent lights providing a 16 hday/8 h night cycle. Cultures are subcultured every two weeks byinoculating approximately 35 mg of tissue into 35 mL of fresh liquidmedia.

In particle gun bombardment procedures it is possible to use purified 1)entire plasmid DNA; or 2) DNA fragments containing only the recombinantDNA expression cassette(s) of interest. For every seventeen bombardmenttransformations, 85 μL of suspension is prepared containing 1 to 90picograms (pg) of plasmid DNA per base pair of each DNA plasmid.Recombinant DNA plasmids are precipitated onto gold particles asfollows. The DNA in suspension is added to 50 μL of a 20-60 mg/mL 0.6 μmgold particle suspension and then combined with 50 μL CaCl₂ (2.5 M) and20 μL spermidine (0.1 M). The mixture is vortexed for 5 sec, spun in amicrofuge for 5 sec, and the supernatant is removed. The DNA-coatedparticles are then washed once with 150 μl of 100% ethanol, vortexed andspun in a microfuge again, then resuspended in 85 μL of anhydrousethanol. Five μL of the DNA-coated gold particles are then loaded oneach macrocarrier disk.

Approximately 150 to 250 mg of two-week-old suspension culture is placedin an empty 60 mm×15 mm petri plate and the residual liquid removed fromthe tissue using a pipette. The tissue is placed about 3.5 inches awayfrom the retaining screen and each plate of tissue is bombarded once.Membrane rupture pressure is set at 650 psi and the chamber is evacuatedto −28 inches of Hg. Three plates are bombarded, and followingbombardment, the tissue from each plate is divided between two flasks,placed back into liquid media, and cultured as described above.

Seven days after bombardment, the liquid medium is exchanged with freshSB 196 medium supplemented with 30-50 mg/L hygromycin. The selectivemedium is subsequently refreshed weekly or biweekly. Seven weekspost-bombardment, bright green, transformed tissue is observed growingfrom untransformed, chlorotic or necrotic embryogenic clusters. Isolatedgreen tissue is removed and inoculated into individual wells in six-wellculture dishes to generate new, clonally-propagated, transformedembryogenic suspension cultures. Thus, each new line is treated as anindependent transformation event in an individual well. Thesesuspensions can then be maintained as suspensions of embryos clusteredin an immature developmental stage through subculture or they can beregenerated into whole plants by maturation and germination ofindividual somatic embryos.

After two weeks in individual cell wells, transformed embryogenicclusters are removed from liquid culture and placed on solidified medium(SB166) containing no hormones or antibiotics for one week. Embryos arecultured at 26° C. with mixed fluorescent and incandescent lights on a16 h day/8 h night schedule. After one week, the cultures are thentransferred to SB 103 medium and maintained in the same growthconditions for 3 additional weeks.

Somatic embryos become suitable for germination after four weeks and arethen removed from the maturation medium and dried in empty petri dishesfor one to five days. The dried embryos are then planted in SB71-4medium where they are allowed to germinate under the same light andtemperature conditions as described above. Germinated embryos aretransferred to sterile soil and grown to maturity for seed production.Oil content and fatty acid composition of the seed are determined asdescribed in previous examples.

Example 10 Transferring Amino Acid Substitutions from Novel HazelnutDGAT into Maize DGAT Increases Specific Activity of Maize DGAT

Six amino acid substitutions frequently observed in novel DGAT sequencesderived from CA-DGAT1* were transferred to maize DGAT to determinewhether these same substitutions could improve the maize enzyme. Themaize DGAT gene used for this example is named ZM-DGAT(MOD1) andcorresponds to SEQ ID NO: 163. This maize DGAT is identical in aminoacid sequence to SEQ ID No: 19 used for sequence homology assessment inTable 2 of EXAMPLE 1. However, the nucleotide sequence of ZM-DGAT(MOD1)was changed to decrease the extremely high GC content in the 5′ regionof the gene to facilitate cloning. The 6 amino acid substitutionstransferred from the novel DGAT genes derived from CA-DGAT1* are L247V,N288D, K356Q, C390S, G451A, and F514S. The corresponding substitutionsin maize DGAT are L201V, N242D, K310Q, C344S, G405A, and F468S. Two ofthe 6 mutations, C344S and F468S, were also made singly and together inmaize DGAT. The names, amino acid substitutions and SEQ ID numbers forthe novel maize DGATs are ZM-DGAT(MOD2), F468S, SEQ ID NO: 163 and 164;ZM-DGAT(MOD3), C344S, SEQ ID NO: 165 and 166; ZM-DGAT(MOD4),F468S/C344S, SEQ ID NO: 167 and 168, and ZM-DGAT(MOD5),L247V/N288D/K356Q/C390S/G451A/F514S, SEQ ID NO: 169 and 170. Theconstruct named PHP40102 comprising the ZM-DGAT(MOD4) gene in the yeastexpression vector is presented as SEQ ID NO: 171 as a representativeexample. Following expression of the novel maize DGATs in theSaccharomyces DGAT/PDAT null, microsomal DGAT activity assays andwestern blots were done. Microsomal membrane preparations and DGATactivity assays were done as described in EXAMPLE 2. The endogenous DAGconcentration and 10 μM 18:1-CoA appeared to be saturatingconcentrations for both of these substrates with all of these maizeenzymes. Western blots were done as described in EXAMPLE 5, except thatthe rabbit polyclonal antibodies used were prepared against the peptideRLRRAPSADAGDLAGD (peptide 2, SEQ ID NO: 177), corresponding to aminoacid positions 27 to 42 of maize DGAT.

Results of the activity assays and western blots are reported in TABLE20. As evident in the right column of TABLE 19, all of the novel maizeDGATs had greater activity than ZM-DGAT(MOD1), following adjustment forDGAT protein abundance. The novel maize DGAT ZM-DGAT(MOD4), whichcontained two amino acid substitutions, had an adjusted activity thatwas more than 3-fold greater than that of ZM-DGAT(MOD1). Novel maizeDGAT ZM-DGAT(MOD2) had about 2.4 fold greater adjusted activity thanZM-DGAT(MOD 1), and the other two novel DGATs had approximately doublethe adjusted activity of ZM-DGAT(MOD1). Such large increases in DGATspecific activity as a result of mutagenesis as observed here for thenovel maize DGATs and in EXAMPLE 5 for the novel hazelnut DGATs have notbeen previously reported. The novel maize DGATs were less abundant inyeast microsomes than was the ZM-DGAT(MOD1) protein. As mentioned inEXAMPLE 5, less abundance may be due to either lower expression level inyeast or due to the fact that proportionately more of the novel DGATproteins were in the fat pad, rather than the microsomal pellet,compared with the ZM-DGAT(MOD1) protein. These results show that aminoacid substitutions present in novel DGAT genes derived from the hazelnutDGAT gene, and identified by screening for high oil content in yeast,can kinetically improve a type 1 DGAT from a different plant. Becausethis transfer of amino acid substitutions was successful going from adicot DGAT to a monocot DGAT with only 64% amino acid sequence identity(TABLE2), it may be expected that transfer of amino acid substitutionsfrom novel DGATs derived from hazelnut DGAT to more closely relateddicot DGATs such as those from the oilseeds soybean (74% identity withhazelnut DGAT, TABLE 2), canola, or sunflower may also be successful.

TABLE 19 Activity and Relative Abundance of Novel Maize DGAT DGAT DGATActivity DGAT Abundance in DGAT Adjusted for Expressed, microsomesActivity¹ DGAT Abundance Amino Acid DGAT relative to Adjusted for (% ofSubstitutions Activity¹ ZM-DGAT(MOD1) DGAT Abundance ZM-DGAT(MOD1)ZM-DGAT(MOD1), 1067 ± 13 1 1067 100 No substitutions ZM-DGAT(MOD2), 1698± 94 0.66 2573 241 F468S ZM-DGAT(MOD3), 1278 ± 75 0.61 2095 196 C344SZM-DGAT(MOD4), 2507 (only 1 0.61 4110 385 F468S/C344S rep done)ZM-DGAT(MOD5), 1288 ± 36 0.62 2077 195 F468S/C344S/ L201V/N242D/K310Q/G405A ¹Activity is measured as pmol C14 labeled oleoyl-CoAincorporated into TAG per minute per mg microsomal protein.

Example 11 Construction of Chimeras Comprising Novel DGAT Genes

The type I DGAT polypeptides from higher plant species have high overallamino acid sequence identities, evident in TABLE 2 of EXAMPLE 1.However, sequence identity is much lower in the N-terminal region(approximately ⅕ of the polypeptide), than it is in the remainder of thepolypeptide. The DGAT N-terminal variable region from a plant speciesmay improve efficacy when ectopically expressing a foreign DGAT obtainedfrom a different plant species. Chimeric genes were constructed thatencode the N-terminal 112 amino acids of maize DGAT fused to theC-terminal 381 amino acids of wild type or novel hazelnut DGAT. The wildtype hazelnut DGAT chimera is named ZM-DGAT:CA-DGAT1, and corresponds toSEQ ID NO: 172 and 173. The novel hazelnut DGAT chimera is namedZM-DGAT:CA-DGAT1-C11, and corresponds to SEQ ID NO: 174 and 175. Thisnovel DGAT chimera is presented as a representative example. Similarchimeras may be made with any of the novel DGAT genes disclosed in thisinvention.

Example 12 Ectopic Expression of Novel DGAT Genes in Maize

Constructs containing an embryo-preferred 16-kD oleosin promoter drivingexpression of novel DGAT genes are transformed into maize byAgrobacterium infection. The constructs also contain a red fluorescentprotein DS-RED driven by an aleurone-specific lipid transfer protein 2(LTP2) promoter to facilitate segregation of transgenic and null kernelsfor phenotypic analysis. Oil data and fatty acid profiles are obtainedfrom transgenic and null kernels.

For Agrobacterium-mediated transformation of maize with novel DGATcDNAs, the method of Zhao is employed (U.S. Pat. No. 5,981,840, and PCTpatent publication WO98/32326; the contents of which are herebyincorporated by reference). Briefly, immature embryos are isolated frommaize and the embryos contacted with a suspension of Agrobacterium,where the bacteria are capable of transferring the DGAT cDNA, operablylinked to a promoter of interest, to at least one cell of at least oneof the immature embryos (step 1: the infection step). In this step theimmature embryos are immersed in an Agrobacterium suspension for theinitiation of inoculation. The embryos are co-cultured for a time withthe Agrobacterium (step 2: the co-cultivation step). The immatureembryos are cultured on solid medium following the infection step.Following this co-cultivation period an optional “resting” step iscontemplated. In this resting step, the embryos are incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). The immature embryos are culturedon solid medium with antibiotic, but without a selecting agent, forelimination of Agrobacterium and for a resting phase for the infectedcells. Next, inoculated embryos are cultured on medium containing aselective agent and growing transformed callus is recovered (step 4: theselection step). The immature embryos are cultured on solid medium witha selective agent resulting in the selective growth of transformedcells. The callus is then regenerated into plants (step 5: theregeneration step), and calli grown on selective medium are cultured onsolid medium to regenerate the plants.

This transformation method was successfully used previously to increaseoil and oleic content in maize by ectopic expression of maize DGAT(Zheng et al. Nature Genetics 40: 367-372). This same method is usedhere to determine whether even greater increases in maize oil or oleiccontents may be achieved through expression of novel DGAT genesdisclosed here. Examples of DGAT genes that are tested with this methodare SEQ ID NO: 163, 165, 167, 169, 11, 27, 49, 51, 53, 172, and 174. TheDGAT genes from dicots may be codon optimized for maize to increaseexpression level. As a representative example, plasmid PHP36707 (FIG. 4)comprises the novel DGAT CA-DGAT1-C11 in the maize transformationvector.

Example 13 Ectopic Expression of Novel DGAT Genes in a High OleicBackground

Oil from hazelnut has greater than 80% oleic acid (Cristofori et al. JSci Food Agric 88:1091-1098). Therefore, the DGAT from this species maybe especially effective in high oleic tissues containing a highproportion of oleoyl-CoA and di-oleoyl diacylglycerol as substratesduring the oil formation period of development. Furthermore, at leastsome of the novel DGATs that give high oil content in yeast may haveimproved affinity (lower Km or S_(0.5) values) for oleoyl-CoA anddi-oleoyl diacylglycerol, as a result of their numerous amino acidsubstitutions. It is therefore of interest to express the novel DGATgenes disclosed here in high oleic plants. Ectopic expression of novelDGAT genes in a high oleic background is achieved by crossing transgenicplant lines ectopically expressing these DGATs with lines having reducedFAD2 (delta-12 fatty acid desaturase) expression. Alternatively,cassettes for DGAT expression and cassettes containing FAD2 inhibitorypolynucleotide sequences can be simultaneously transformed by themethods of Examples 10 and 11. Examples of FAD2 inhibitorypolynucleotide sequences and inhibitory constructs for maize include,but are not limited to, those disclosed in U.S. Patent ApplicationPublication No. 2005-0160494 and WO 2005/063988, herein incorporated byreference in their entirety.

Example 14 Testing Novel Hazelnut DGAT Genes from Library J in SoybeanSomatic Embryos and Transferring Library J Amino Acid Substitutions intoSoybean DGAT

Four novel DGAT genes (CA-DGAT1-J16, CA-DGAT1-J24, CA-DGAT1-J32, andCA-DGAT1-J37) that gave high oil when expressed in yeast, plus theCA-DGAT1* gene, are ectopically expressed in soybean somatic embryosunder control of the soybean β-conglycinin α′ subunit promoter using themethods of EXAMPLE 3. Oil content and fatty acid profiles of soybeansomatic embryos are determined as described herein. It is likely thatsome or all of the novel DGAT genes from library J will give rise tohigher oil and oleic acid concentrations in somatic embryos compared toCA-DGAT1*. Similar results will be observed for these genes whenexpressed in seed using techniques described herein.

Some or all of the amino acid substitutions present in these novel hazelDGATs from library J will be transferred to soybean DGAT to determinewhether these same substitutions can increase the effectiveness ofsoybean DGAT for increasing oil or oleic content. The substitutions thatare transferred are summarized in TABLE 20. The names as well as DNA anddeduced amino acid sequences for the five novel soybean DGAT genes areGM-DGAT1-J16 (SEQ ID NO:184 and 185), GM-DGAT1-J24 (SEQ ID NO:186 and187), GM-DGAT1-J32 (SEQ ID NO:188 and 189), DGAT1-J37 (SEQ ID NO:190 and191) and GM-DGAT1-J16J24J32J37 (SEQ ID NO:192 and 193). These five novelsoybean DGAT proteins contained 11, 11, 11, 9 and 15 amino acidsubstitutions, respectively, A DNA and deduced amino acid sequenceincorporating all of the mutations described in Example 8 from library Cand here from library J (GM-DGAT1-Jall Call) are set forth in SEQ IDNO:194 and SEQ ID NO:195, respectively.

TABLE 20 Amino Acid Substitutions in Novel Hazelnut DGATs and theCorresponding Substitutions Made in Soybean DGATs Hazelnut¹ (J16, J24,Soy Soy Soy Soy Soy² J32, or J37) (J16) (J24) (J32) (J37) (J16J24J32J37)N31A S24A S24A S24A S24A S181A S146A S146A S146A R241K R206K R206K R206KR206K R206K T251V T216V T216V T216V T216V Y266F Y231F Y231F Y231F S299TS264T S264T S264T V308L V273L V273L V273L V273L V273L V334L V299L V299LV299L I338V I303V I303V I303V C390S C355S C355S C355S C355S C355S L399VL364V L364V L364V L364V L364V V436R I401R i401R I475M I440M I440M I440MI440M F514S I479S I479S I479S I479S L518V L483V L483V L483V L483V ¹Somesubstitutions observed in novel hazelnut DGAT genes CA-DGAT1-J16,CA-DGAT1-J24, CA-DGAT1-J32 or CA-DGAT1-J37; SEQ ID NOs: 138, 142, 144,148, respectively. ²SEQ ID NO: 193, incorporates all of the changes fromGM-DGAT1-J16, GM-DGAT1-J24, GM-DGAT1-J32, and GM-DGAT1-J37; SEQ ID NOs:185, 187, 189, and 191, respectively.

Example 15 Analysis of Kernel Oil Content Nuclear Magnetic Resonance(NMR) Analysis:

Seed are imbibed in distilled water for 12-24 hours at 4° C. The embryois dissected away and stored in a 48 well plate. The samples arelyophilized over-night in a Virtis 24×48 lyophilizer. The NMR (ProcessControl Technologies—PCT (Ft. Collins, Colo.) is set up as per themanufacturer's instructions. The NMR is calibrated using a series of 5mm NMR tubes containing precisely measured amounts of corn oil (Mazola).The calibration standards are 3, 6, 9, 12, 15, 18, 21, 27, 33, and 40 mgof oil.

Example 16 Compositional Analysis of Soybean Seed

The present example describes measurements of seed compositionalparameters such as protein content and content of soluble carbohydratesof soybean seed derived from transgenic events that express DGAT genes.

Changes in the composition of soybean seed associated with expression ofDGAT genes are measured. To this end the concentrations of protein,soluble carbohydrates and starch are measured as follows.

Non-Structural Carbohydrate and Protein Analysis.

Dry soybean seed are ground to a fine powder in a GenoGrinder andsubsamples are weighed (to an accuracy of 0.1 mg) into 13×100 mm glasstubes; the tubes have Teflon® lined screw-cap closures. Three replicatesare prepared for each sample tested. Tissue dry weights are calculatedby weighing sub-samples before and after drying in a forced air oven for18 h at 105 C.

Lipid extraction is performed by adding 2 ml aliquots of heptane to eachtube. The tubes are vortex mixed and placed into an ultrasonic bath (VWRScientific Model 750D) filled with water heated to 6° C. The samples aresonicated at full-power (˜360 W) for 15 min and are then centrifuged (5min×1700 g). The supernatants are transferred to clean 13×100 mm glasstubes and the pellets are extracted 2 more times with heptane (2 ml,second extraction, 1 ml third extraction) with the supernatants fromeach extraction being pooled. After lipid extraction 1 ml acetone isadded to the pellets and after vortex mixing, to fully disperse thematerial, they are taken to dryness in a Speedvac.

Non-Structural Carbohydrate Extraction and Analysis.

Two ml of 80% ethanol is added to the dried pellets from above. Thesamples are thoroughly vortex mixed until the plant material is fullydispersed in the solvent prior to sonication at 60 C for 15 min. Aftercentrifugation, 5 min×1700 g, the supernatants are decanted into clean13×100 mm glass tubes. Two more extractions with 80% ethanol areperformed and the supernatants from each are pooled. The extractedpellets are suspended in acetone and dried (as above). An internalstandard β-phenyl glucopyranoside (100 ul of a 0.5000+/−0.0010 g/100 mlstock) is added to each extract prior to drying in a Speedvac. Theextracts are maintained in a desiccator until further analysis.

The acetone dried powders from above are suspended in 0.9 ml MOPS(3-N[Morpholino]propane-sulfonic acid; 50 mM, 5 mM CaCl₂, pH 7.0) buffercontaining 100U of heat stable α-amylase (from Bacillus licheniformis;Sigma A-4551). Samples are placed in a heat block (90 C) for 75 min andare vortex mixed every 15 min. Samples are then allowed to cool to roomtemperature and 0.6 ml acetate buffer (285 mM, pH 4.5) containing 5Uamyloglucosidase (Roche 110 202 367 001) is added to each. Samples areincubated for 15-18 h at 55 C in a water bath fitted with areciprocating shaker; standards of soluble potato starch (Sigma S-2630)are included to ensure that starch digestion goes to completion.

Post-digestion the released carbohydrates are extracted prior toanalysis. Absolute ethanol (6 ml) is added to each tube and after vortexmixing the samples are sonicated for 15 min at 60 C. Samples arecentrifuged (5 min×1700 g) and the supernatants are decanted into clean13×100 mm glass tubes. The pellets are extracted 2 more times with 3 mlof 80% ethanol and the resulting supernatants are pooled. Internalstandard (100 ul β-phenyl glucopyranoside, as above) is added to eachsample prior to drying in a Speedvac.

Sample Preparation and Analysis

The dried samples from the soluble and starch extractions describedabove are solubilized in anhydrous pyridine (Sigma-Aldrich P57506)containing 30 mg/ml of hydroxylamine HCl (Sigma-Aldrich 159417). Samplesare placed on an orbital shaker (300 rpm) overnight and are then heatedfor 1 hr (75 C) with vigorous vortex mixing applied every 15 min. Aftercooling to room temperature 1 ml hexamethyldisilazane (Sigma-AldrichH-4875) and 100 ul trifluoroacetic acid (Sigma-Aldrich T-6508) areadded. The samples are vortex mixed and the precipitates are allowed tosettle prior to transferring the supernatants to GC sample vials.

Samples are analyzed on an Agi lent 6890 gas chromatograph fitted with aDB-17MS capillary column (15 m×0.32 mm×0.25 um film). Inlet and detectortemperatures are both 275 C. After injection (2 ul, 20:1 split) theinitial column temperature (150 C) is increased to 180 C at a rate 3C/min and then at 25 C/min to a final temperature of 320 C. The finaltemperature is maintained for 10 min. The carrier gas is H₂ at a linearvelocity of 51 cm/sec. Detection is by flame ionization. Data analysisis performed using Agilent ChemStation software. Each sugar isquantified relative to the internal standard and detector responses areapplied for each individual carbohydrate (calculated from standards runwith each set of samples). Final carbohydrate concentrations areexpressed on a tissue dry weight basis.

Protein Analysis

Protein contents are estimated by combustion analysis on a ThermoFinnigan Flash 1112EA combustion analyzer. Samples, 4-8 mg, weighed toan accuracy of 0.001 mg on a Mettler-Toledo MX5 micro balance are usedfor analysis. Protein contents are calculated by multiplying % N,determined by the analyzer, by 6.25. Final protein contents areexpressed on a % tissue dry weight basis.

Example 17 Analysis of Lipid Fractions of Transgenic Seed and SomaticEmbryos Expressing DGAT Genes Total Lipid Extraction

Total lipid is extracted from each event by the method of Bligh, E. G. &Dyer, W. J. (Can. J. Biochem. Physiol. 37:911-917 (1959)) with somemodifications. Briefly, approximately 100 mg of ground tissue from eachevent is added to a 16 mm×125 mm sized test-tube with a teflon-linedscrew cap lid. A mixture of methanol:chloroform/2:1 (6 mL) is added andthe sample is extracted with gentle mixing for 1 hr after which 2 mL ofchloroform is added followed by continued mixing for 30 min. Afterwards,3.6 mL of water is added, the tube is vortexed vigorously and phases areseparated by centrifugation in a clinical centrifuge. The lower organiclayer is gently removed to a second glass test tube and the upperaqueous layers are re-extracted with 2 mL of chloroform. Centrifugationis repeated and the lower organic phase is combined with the firstorganic phase. Samples are dried under a stream of nitrogen at 50 C,total lipid is estimated by weighing and lipid is dissolved inchloroform:methanol/6:1 to a concentration of approximately 10 mg/mL.FAME analysis is carried out on approximately 50 ug of each sample usingthe sulfuric acid/methanol procedure described herein (Example 4) andresults are shown in Table 30.

Separation of Neutral and Polar Lipids

Sep-pak amino-propyl solid phase extraction columns (Waters; 6 cccolumns, WAT054560) are equilibrated with 5 mL of methanol followed by 5mL of methanol:chloroform/1:1 followed by 5 mL of chloroform.Approximately 5 mg of total lipid in chloroform:methanol/6:1 is added toeach column, followed by 5×1 mL aliquots of chloroform to elute neutrallipids and all fractions are collected, combined and dried under astream of nitrogen at 50 C. Polar lipids are then eluted from eachcolumn using 5×1 mL aliquots of methanol:chloroform/1:1 followed by 5×1mL aliquots of methanol and all fractions are combined and dried undernitrogen. Neutral lipids are dissolved in approximately 1 mL ofCHCl3:MeOH/6:1 and polar lipids are dissolved in approximately 200 uL ofCHCl3:MeOH/6:1. FAME analysis is carried out on approximately 50 ug ofneutral lipid using the sulfuric acid/methanol procedure describedherein (Example 4).

Separation of TAG, PC and PE by TLC

Approximately 100 uL of neutral lipid extract is loaded 2 cm from thebottom of a Partisil K6 Silica Gel 60 A TLC plate (Whatman, 250 umthickness, 20 cm×20 cm). Similarly, approximately 200 uL of the polarlipid fraction is loaded onto the same TLC plate. Standard solutions (10mg/mL in chloroform:methanol/6:1) of TAG, PC and PE are also spottedonto the plates. TLC plates are developed in CHCl3:MeOH:AcOH/65:35:8until solvent front is approximately half way up the plate. TLC platesare then air dried for 10 min and developed fully in 70:30:1 (v/v/v)hexane:diethylether:acetic acid. Standards are visualized by lightstaining with iodine vapour and corresponding bands for TAG, PC and PEare cut out of the TLC plate. Silica gel containing each lipid speciesis derivatized directly with sulfuric acid/methanol as described herein(Example 4) and results are shown in Table 30.

Fatty acid Positional analysis of TAG

Fatty acid profiles of the sn2 position of TAG are determined usingporcine pancreatic lipase to remove acyl groups from the sn1 and sn3position of TAG only, followed by transesterification of the resultingmonoacylglyceride (MAG) produced. Approximately 5 mg of neutral lipidextract is suspended in 2 mL of 1M Tris.HCl, pH 8.0 along with 0.2 mL of2.2% calcium chloride and 0.5 mL of 0.05% Bile salts in a glass screwcap test tube. The lipid is incubated at 37 C for 5 min, 5 mg of porcinepancreatic lipase is added directly and the suspension is incubated withshaking at 37 C for 20 min. After incubation, the reaction is terminatedwith the addition of 1 mL of ethanol followed by 1 mL of 6 M HCl. Aftermixing, 2.5 mL of diethyl ether is added, phases are separated bycentrifugation and the top organic layer is removed carefully. Thediethyl ether extraction is repeated and the top diethyl ether phase iscombined with the first. After drying over anhydrous sodium sulfate, thediethyl ether is evaporated under a stream of nitrogen at 50 C and theresulting lipid is dissolved in 200 uL of chloroform:methanol/6:1. Thelipid is loaded onto a Partisil K6 TLC plate along with triacylglyceride(TAG), diacylglyceride (DAG), monoacylglyceride (MAG) and free fattyacid (FFA) standards and the TLC plate is developed as described herein.Afterwards, standards are visualized with light iodine staining and theMAG band is cut and derivatized with methanol/sulfuric acid aspreviously described herein. The % of total fatty acid for each fattyacid (i.e. 16:0, 18:0, 18:1, 18:2, 18:3) at the sn1 and sn3 positions ofTAG is calculated with the following formula: =([TAGx]−[sn2x]/3)* 3/2;where the x indicates the fatty acid of interest.

What is claimed is:
 1. An isolated polynucleotide comprising: (a) anucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity wherein the polypeptide has at least 80% aminoacid identity, based on the Clustal V method of alignment, when comparedto an amino acid sequence as set forth in SEQ ID NOs:8, 10, or 12; (b) anucleotide sequence encoding a polypeptide having diacylglycerolacyltransferase activity, wherein the nucleotide sequence has at least80% sequence identity, based on the BLASTN method of alignment, whencompared to a nucleotide sequence as set forth in SEQ ID NO: 7, 9, or11: (c) a nucleotide sequence encoding a polypeptide havingdiacylglycerol acyltransferase activity, wherein the nucleotide sequencehybridizes under stringent conditions to a nucleotide sequence as setforth in SEQ ID NO: 7, 9, or 11; or (d) a complement of the nucleotidesequence of (a), (b) or (c), wherein the complement and the nucleotidesequence consist of the same number of nucleotides and are 100%complementary.